Laser Microdissection of Paraffin Embedded Tissues ...

JUSTUS-LIEBIG-UNIVERSITY GIESSEN Faculty 09 – Agricultural Sciences, Nutritional Sciences, and Environmental Management

Institute for Nutritional Sciences – Human Nutrition – Physiological Evaluation of Food – Prof. Dr. Clemens Kunz

Master’s Thesis Laser Microdissection of Paraffin Embedded Tissues to Analyze N-Glycan Structures

Submitted by Ashkan Madani

Study Course:

Nutritional Sciences (Master of Science)

Reference Module:

MK 42 Nutrition and Metabolism

Advisor:

PD Dr. Sebastian Galuska

First Supervisor:

Prof. Dr. Clemens Kunz

Second Supervisor: PD Dr. Sebastian Galuska

Giessen, 30th May 2017

Acknowledgements

Acknowledgements First, I would like to thank my thesis advisor PD Dr. Sebastian Galuska of the Leibniz-Institute for Farm Animal Biology at the Institute of Reproductive Biology, Glycobiology Group. The door to PD Dr. Sebastian Galuska office was always open whenever I needed him. He had always time for me no matter if it was a simple question about my research, a trouble spot or even personal conversations. He steered me in the right direction, but I was always allowed to work independently and implement new ideas. I received much trust and he has always believed in a successful outcome, even after so many failed trials. He took always his time and was without hesitation present in the lab when I needed him. I am deeply thankful to you and I appreciate your patience, trust and his scientific council. I cannot remember a single day in day lab without having fun at work. Being part of your group was a pleasure. I want to thank Dr. Christina Galuska for teaching me techniques like permethylation or sample purification via SPE. And mostly, thank you for teaching me “how to troubleshoot a leaking HPLC system”. I really felt like a professional when I was able to fix the HPLC system on my own. Dr. Christina Galuska had likewise a sympathetic ear for every question I had and I was really lucky to have you a second experienced and professional advisor for my thesis. Beside my advisors, I would like to thank the whole Glycobiology Group. First, Farhad Khosravi for introducing me to the first experiments and availability when I needed help. I want also to thank my fellow lab mates, Farzali Husejnov, Natalia Panasenko, Jan Dambon, Philipp Christian, Andrea Kühne, and Kristina Zlatina. We struggled together, we helped each other, and I had the best time with you. The good atmosphere helped in our lab always helped me to keep up in hard times. Much gratitude goes to all laboratory technical assistant, especially Petra Reckling and Gesine Krüger. I cannot tell how much I learned from you and how great your service was. There is not possible way to appreciate everything I got from you. Furthermore, I want to thank Dr. Marten Michaelis for questioning all my methodological approaches leading me to rethink my protocols. Certainly, questioning consisting approaches is a very important scientific tool. My thanks go also to Dr. Elke Albrecht for assisting me at the Laser Microdissection and giving me helpful instructions. All in all, I had a really good time at the Leibniz Institute for Farm Animal Biology. I had the honor to meet great minds, coworkers and people. I am also grateful to Dr. Michaela Hohberger for her medical advice and treatment in times of need. Benjamin Laufer, Beata Berlin, Daniel Glatzel and Felix Klöpping made me a huge favor by reading my manuscript from different point of views. It was a huge help to me. I want to thank Prof. Dr. Clemens Kunz for giving me the permission to perform my thesis outside our faculty without hesitation and encouraging me to experience new scientific insights. I am so grateful to my parents after an intensive period of time during my studies of nutritional sciences. Your wise counsel and sympathetic ear was always helpful. You convinced me eight years ago to leave the army and to start studying and I have to admit that this was the best decision I made so far. I received great education at the Justus-Liebig University Giessen, met great fellow students and had great professors and post-doctoral researchers as teachers.

I

Declaration of Authorship

Declaration of Authorship

I hereby declare that the thesis submitted is my own unaided work. All direct or indirect sources used in this work are acknowledged as references and I have not used any sources other than specified.

I am aware that the thesis can be examined by plagiarism software in order to determine whether the thesis as a whole or parts incorporated in it may be deemed as plagiarism. For the comparison of my work with existing sources I agree that it shall be entered in a database where it shall also remain after examination, to enable comparison with future theses submitted.

This work was not previously presented to another examination board and has not been published.

Ashkan Madani Fasanenweg 21 60437 Frankfurt am Main (Germany born 16th April 1986 in Tehran (Iran) [email protected] [email protected]

Frankfurt am Main, 30th May 2017 (Place, Date)

(Personal signature) II

List of Contents

List of Contents Acknowledgements ................................................................................................................. I Declaration of Authorship ....................................................................................................... II List of Contents ..................................................................................................................... III List of Figures ...................................................................................................................... VII List of Tables ........................................................................................................................ IX List of Abbreviations .............................................................................................................. X 1

Introduction ...................................................................................................................... 1 1.1

Glycobiology ............................................................................................................ 1

1.2

General Biological Function of Glycans ................................................................... 3

1.2.1 Glycosidic Bonds and Linkages.......................................................................... 4 1.2.2 Monosaccharide Components of Mammalian Glycans ....................................... 5 1.2.3 Glycans in Health and Disease .......................................................................... 6 1.2.4 Glycans in Clinical Use ...................................................................................... 7 1.3

Protein Glycosylation ............................................................................................... 8

1.3.1 N-Glycans ........................................................................................................ 10 1.3.2 O-Glycans ........................................................................................................ 11 1.4

Synthesis of N-Glycans ......................................................................................... 12

1.4.1 Synthesis of N-Glycan Precursor in the Rough Endoplasmic Reticulum........... 12 1.4.2 N-Glycan Synthesis in the Golgi-Apparatus ..................................................... 14 2

Methodological Principles............................................................................................... 16 2.1

Aim and Hypothesis of this Work ........................................................................... 16

2.2

Standards .............................................................................................................. 18

2.2.1 Dextran ............................................................................................................ 18 2.2.2 Fetuin and Asialofetuin ..................................................................................... 18 2.2.3 Ribonuclease B ................................................................................................ 18 2.2.4 Human Transferrin ........................................................................................... 18 2.3

Formalin-Fixed Paraffin Embedded Tissue ............................................................ 19

2.3.1 General information.......................................................................................... 19 2.3.2 Deparaffinization .............................................................................................. 19 2.3.3 Antigen retrieval ............................................................................................... 19 2.4

Laser Capture Microdissection .............................................................................. 20

2.4.1 History.............................................................................................................. 20 2.4.2 Principles of Laser Capture Microdissection ..................................................... 21 2.4.3 Laser Capture Microdissection Techniques ...................................................... 21 2.5

N-Glycan Processing ............................................................................................. 24

2.5.1 Enzymatic N-Glycan Release ........................................................................... 24 2.5.2 Fluorescence Labeling of N-Glycans ................................................................ 25 III

List of Contents

2.5.3 Purification of Excessive Labeling Reagents .................................................... 26 2.6

High Performance Liquid Chromatography ............................................................ 27

2.7

Permethylation ....................................................................................................... 28

2.8

Mass Spectrometry ................................................................................................ 29

2.8.1 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry ..................... 30 2.8.2 Electrospray-Ionization Mass Spectrometry ..................................................... 30 2.8.3 Fragmentation of Glycans ................................................................................ 31 2.8.4 Exoglycosidase Digestion ................................................................................ 31 3

Methods ......................................................................................................................... 32 3.1

Preparation of Standard Glycans ........................................................................... 32

3.1.1 Preparation of Dextran Hydrolysate ................................................................. 32 3.1.2 Preparation of Standard Glycoproteins............................................................. 32 3.2

Preparation of Formalin-Fixed Paraffin Embedded Tissues ................................... 32

3.2.1 Cutting Procedure ............................................................................................ 33 3.2.2 Deparaffinization and Staining on PEN and Histology Glass Slides ................. 34 3.2.3 Deparaffinization in Tubes................................................................................ 35 3.3

Laser Capture Microdissection .............................................................................. 35

3.4

Antigen retrieval ..................................................................................................... 39

3.4.1 Trypsin Mediated Antigen Retrieval .................................................................. 39 3.4.2 Heat Mediated Antigen Retrieval ...................................................................... 40 3.5

N-Glycan Release, Purification and Derivatization ................................................. 42

3.5.1 N-Glycan Release from Dot-Blotted Glycoproteins onto PVDF Membranes ..... 42 3.5.2 N-Glycan Release in Solution .......................................................................... 44 3.5.3 N-Glycan Purification ....................................................................................... 44 3.5.4 Derivatization with 2-Aminobenzamide............................................................. 45 3.5.5 Purification of N-Glycans from Excess 2-Aminobenzamide .............................. 46 3.6

High Performance Liquid Chromatography ............................................................ 48

3.6.1 Column I: TSKgel® Amide-80 Å, 4.6 mm x 25.0 cm .......................................... 49 3.6.2 Column II: SeQuant® ZIC®-HILIC, PEEK Coated, 200 Å, 150 x 4.6 mm ........... 50 3.6.3 Column III: SeQuant® ZIC®-HILIC, PEEK Coated, 100 Å, 250 x 2.1 mm .......... 51

4

3.7

Permethylation ....................................................................................................... 51

3.8

Mass Spectrometry ................................................................................................ 52

Results ........................................................................................................................... 53 4.1

Establishment of an HPLC Protocol ....................................................................... 53

4.1.1 Dextran Ladder ................................................................................................ 53 4.1.2 Standard Glycoproteins: Trypsin Mediated Antigen Retrieval ........................... 54 4.1.3 Standard Glycoproteins: Heat Mediated Antigen Retrieval ............................... 55 4.1.4 Limit of Detection ............................................................................................. 57 4.2

N-Glycan Analysis of Whole FFPE Tissue Sections............................................... 59 IV

List of Contents

4.3

N-Glycan Analysis of Laser Microdissection Samples ............................................ 61

4.4

Purification of Excess 2-Aminobenzamide Label Reagent ..................................... 66

4.4.1 Hydrophilic Liquid Interaction Chromatography Solid Phase Extraction............ 66 4.4.2 Isocratic Separation Prior Gradient Separation ................................................ 67 4.5 5

Mass Spectrometry Analysis of Permethylated N-Glycans..................................... 68

Discussion ..................................................................................................................... 70 5.1

Establishment of an HPLC Protocol ....................................................................... 70

5.2

N-Glycan Analysis of Whole FFPE Tissue Sections............................................... 71

5.3

N-Glycan Analysis from Laser Microdissected Cells .............................................. 72

5.4

Analysis of Permethylated N-Glycans through Mass Spectrometry ........................ 73

5.5

Conclusion and Future Perspectives ..................................................................... 74

6

Abstract.......................................................................................................................... 76

7

References ....................................................................................................................... i 7.1

8

Internet Links .......................................................................................................... xii

Appendix ........................................................................................................................ xiii 8.1

Materials ................................................................................................................ xiii

8.1.1 Preparation of Dextran Hydrolysate ................................................................. xiii 8.1.2 Preparation of Standard Glycoproteins............................................................. xiii 8.1.3 Cutting Procedure ............................................................................................ xiv 8.1.4 Deparaffinization and Staining on PEN and Histology Glass Slides ................. xiv 8.1.5 Deparaffinization in Tubes................................................................................ xv 8.1.6 Laser Capture Microdissection ......................................................................... xv 8.1.7 Trypsin Mediated Antigen Retrieval .................................................................. xvi 8.1.8 Heat Mediated Antigen Retrieval ...................................................................... xvi 8.1.9 N-Glycan Release from Dot-Blotted Glycoproteins onto PVDF Membranes .... xvii 8.1.10 N-Glycan Release in Solution ......................................................................... xvii 8.1.11 N-Glycan Purification ..................................................................................... xviii 8.1.12 Derivatization with 2-Aminobenzamide........................................................... xviii 8.1.13 Purification of N-Glycans from Excess 2-Aminobenzamide .............................. xix 8.1.14 High Performance Liquid Chromatography ...................................................... xx 8.1.15 Permethylation ................................................................................................. xxi 8.1.16 Mass Spectrometry ......................................................................................... xxii 8.2

Additional Figures ................................................................................................. xxii

8.3

Additional Tables ................................................................................................. xxiv

8.4

Additional Protocols ............................................................................................. xxxi

8.4.1 Purification of Permethylated N-Glycans ........................................................ xxxi 8.4.2 Cleaning of the Stainless Steel AnchorChip Target ........................................ xxxi 8.4.3 Esterification of Linkage Specific Esterification of Sialic Acids ........................ xxxi 8.5

Calculations ........................................................................................................ xxxii V

List of Contents

8.5.1 Trifluoroacetic Acid (0.1 M) ........................................................................... xxxii 8.5.2 Ammoniums Bicarbonate (25 mM, pH 8.0) .................................................... xxxii 8.5.3 Trypsin (1 mg mL−1) in 0.001 N Hydrochloric Acid ......................................... xxxii 8.5.4 Phenylmethylsulfonylfluoride (100 mM) ......................................................... xxxii 8.5.5 Tris-Hydrochloride (0.1 M, pH 8) Containing 4% Sodium Dodecyl Sulfate .... xxxiii 8.5.6 Dithiothreitol (0.1 M) ..................................................................................... xxxiii 8.5.7 rea (8 M) ...................................................................................................... xxxiii 8.5.8 Stain Solution ............................................................................................... xxxiii 8.5.9 Destain Solutions ......................................................................................... xxxiv 8.5.10 Polyvinylpyrrolidone 40 0.1% (w/v) ............................................................... xxxiv 8.5.11 2-Aminobenzamide Label............................................................................. xxxiv 8.5.12 Ammonium Formate (50 mM, pH 4.4) ........................................................... xxxv 8.5.13 Ammonium Acetate (5 mM, pH 6.4) .............................................................. xxxv

VI

List of Figures

List of Figures Fig 1: Glycocalyx of an Erythrocyte. ....................................................................................... 1 Fig 2: Association of Glycomics and Other “-Omics” Studies. ................................................ 2 Fig 3: Interaction of Glycoconjugates with Cellular Processes. .............................................. 3 Fig 4: Main Roles of Glycans in Glycoprotein Functions. ....................................................... 4 Fig 5: Brief Summary of Glycosidic Bondages and Branching Points. .................................... 5 Fig 6: Two Most Common Neu5Ac Linkages in Mammals. .................................................... 6 Fig 7: Changes in Protein Glycosylation during Cellular Transformation and Progression. .... 7 Fig 8: Glycoforms. .................................................................................................................. 8 Fig 9: Major Classes of Mammalian Glycan Structures. ....................................................... 10 Fig 10: Types of N-Glycans Added to Polypeptides at Asn-X-Ser/Thr Sequons. .................. 11 Fig 11: Structure of Dolichol Phosphate. .............................................................................. 12 Fig 12: Topological Model of N-Glycan Synthesis in the rER. .............................................. 13 Fig 13: Glycan Precursor attached to LLO or N-Glycosylation Site. ..................................... 14 Fig 14: Glycan Processing in the Golgi Apparatus. .............................................................. 15 Fig 15: Methodological Concept of this Work. ...................................................................... 17 Fig 16: Timeline of Laser Microdissection Technology Development. .................................. 21 Fig 17: Schematic Representation of IR Laser (left) and UV Laser System (right). .............. 22 Fig 18: LCM system PALM Microbeam (Zeiss). ................................................................... 23 Fig 19: N-Glycan Release Catalyzed by PNGase F. ............................................................ 25 Fig 20: Reaction of Reductive Animation by Fluorescence Tags. ......................................... 26 Fig 21: Combination of Different Chromatography Characteristics in HILIC. ........................ 28 Fig 22: Reaction of Permethylation. ..................................................................................... 29 Fig 23: Used Microtome for Tissue Sectioning. .................................................................... 33 Fig 24: Used LCM Device in this Work. ................................................................................ 36 Fig 25: PALM Robo Software 4.5 Pro Surface. .................................................................... 37 Fig 26: Schematic Representation of LCM Protocol. ............................................................ 38 Fig 27: Preparational Steps for Heat Mediated Antigen Retrieval. ........................................ 41 Fig 28: Dot-Blotted Glycoproteins. ....................................................................................... 43 Fig 29: N-Glycan Purification Using Multichannel Pipette and 96-Well Plate. ....................... 45 Fig 30: Preparation of 100% Cotton Wool served as HILIC SPE Microtip. ........................... 46 Fig 31: N-Glycan Purification of Excessive 2AB Using Multichannel Pipette and 96-WP...... 47 Fig 32: HPLC System Used in this Work. ............................................................................. 48 Fig 33: Dextran Ladder Separated by Three Different Columns. .......................................... 53 Fig 34: 2AB Labeled N-Glycan Peaks of Tryptic Digested Standard Glycoproteins. ............. 55 Fig 35: 2AB Labeled N-Glycan Peaks of Human Transferrin................................................ 56 Fig 36: LOD for 2AB Labeled N-Glycans of RNase B Separated with Column II. ................. 57 Fig 37: LOD for 2AB Labeled N-Glycans of RNase B Separated with Column III. ................ 58 Fig 38: LOD for 2AB Labeled N-Glycans of TF after Heat Mediated Antigen Retrieval......... 59 Fig 39: Analysis of 2AB Labeled N-Glycans from Whole FFPE Tissue Sections. ................. 60

VII

List of Figures

Fig 40: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Adult Mouse). ............................................................................................................................................ 61 Fig 41: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Adult Rat). . 62 Fig 42: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Roe Deer Deceased in August 2001). .................................................................................................. 64 Fig 43: Laser Capture Microdissected Cells of Epithelial Cells from Ductus Epididymis. ...... 65 Fig 44: Purification of 2AB Labeled N-Glycans with HILIC-SPE. .......................................... 66 Fig 45: Purification of Excessive 2AB Label Through Isocratic Separation. .......................... 68 Fig 46: Mass Spectrometry Analysis of Permethylated N-Glycans. ...................................... 69 Fig 47: Six-Port High-Pressure Valve with Trap Column for On-Line SPE. .......................... 72 Fig 48: Ethyl Esterification and Lactone Formation of Sialic Acids. ...................................... 73 Fig 49: Nomenclature for Carbohydrate Fragments. ........................................................... xxii Fig 50: Laser Capture Microdissected Sperms from Ductus Epididymis (Roe Deer Deceased in August 2001).................................................................................................................. xxiii

VIII

List of Tables

List of Tables Tab 1: Gradient Separation with HILIC-Column I. ................................................................ 49 Tab 2: Gradient Separation with HILIC-Column II. ............................................................... 50 Tab 3: Gradient Separation with HILIC-Column III. .............................................................. 51 Tab 4: Use of Isocratic Separation at the Beginning to Purify N-Glycans from Excessive 2AB Label (HILIC-Column II). ...................................................................................................... 67 Tab 5: Materials Required for Dextran Hydrolysation. .......................................................... xiii Tab 6: Materials Required for Preparation of Standard Glycoproteins .................................. xiii Tab 7: Material List for Cutting Procedure via Microtome. .................................................... xiv Tab 8: Material List for Deparaffinization and Staining on Histology Glass Slides. ............... xiv Tab 9: Material List for Deparaffinization in Tubes. .............................................................. xv Tab 10: Material List for Laser Capture Microdissection. ...................................................... xv Tab 11: Material List for Trypsin Mediated Antigen Retrieval. .............................................. xvi Tab 12: Material List for Heat Mediated Antigen Retrieval.................................................... xvi Tab 13: Material List for Dot-Blotting of (Glyco)Proteins. ..................................................... xvii Tab 14: Material List for N-Glycan Release in Solution. ...................................................... xvii Tab 15: Material List for N-Glycan Purification. .................................................................. xviii Tab 16: Material List for Labeling with 2-Aminobenzamide. ............................................... xviii Tab 17: Material List for Purification of Excess 2-Aminobenzamide. .................................... xix Tab 18: Material List for HPLC Analysis. .............................................................................. xx Tab 19: Material List for Permethylation of N-Glycans. ........................................................ xxi Tab 20: Material List for MALDI-TOF-MS. ........................................................................... xxii Tab 21: Consensus Motifs and Glycan Linkages. .............................................................. xxiv Tab 22: Mammalian Glycan Linkages produced by Glycosyltransferases. .......................... xxv Tab 23: Expected N-Glycans from Bovine Fetuin B. .......................................................... xxvi Tab 24: Expected N-Glycans from Ribonuclease B. ......................................................... xxvii Tab 25: Expected N-Glycans from Human Transferrin. .................................................... xxviii Tab 26: Examples of Monosaccharide Masses. ................................................................. xxix Tab 27: Common Encountered B-Type Ions. ..................................................................... xxix Tab 28: Residue Masses of Common Terminal Groups. ..................................................... xxx Tab 29: Commonly Used Exoglycosidases and Incubate Conditions. ................................. xxx

IX

List of Abbreviations

List of Abbreviations 2,5-dihydroxybenzoic acid

2,5-DHB

2-aminobenzamide

2AB

2-aminobenzoic acid

2AA

acetonitrile

ACN

amino acid

AA

ammonium acetate

NH4CH3CO2

ammonium bicarbonate

NH4HCO3

ammonium formate

NH4HCO2

arginine

Arg

asparagine

Asn

chloroform/trichloromethane

CHCl3

congenital disorder of glycosylation

CDG

cysteine

Cys

deoxyribonucleic acid

DNA

dichloromethane

CH2Cl2

dimethyl sulfoxide

DMSO

dithiothreitol

DTT

dolichol / dolichol-phosphate / dolichol-pyrophosphate

Dol / Dol-P / Dol-PP

electrospray ionization mass spectrometry

ESI-MS

endoplasmic reticulum associated degradation

ERAD

epidermal growth factor

EGF

erythropoietin

EPO

formalin-fixed paraffin embedded

FFPE

fucose / fucosyl-transferase

Fuc / Fuc-T

galactose / galactosyl-transferase

Gal / Gal-T

glucose

Glc

glycine

Gly

glycosaminoglycan

GAG

Hemalaun & eosin

H&E

high performance liquid chromatography

HPLC

high pH anion exchange chromatography with pulsed amperometric detection

HPAEC-PAD

hydrophilic interaction liquid chromatography

HILIC

hydrochloric acid

HCl

hydroxylysine

Hyl

immunohistochemistry

IHC

infrared

IR

laser capture microdissection

LCM

laser pressure catapulting

LPC

limit of detection

LOD

X

List of Abbreviations

lysine

Lys

mannose

Man

mass spectrometry

MS

mass-to-charge ratio

m/z

matrix-assisted laser desorption/ionization time of flight mass spectrometry

MALDI-TOF-MS

methyl iodide

CH3I

N-acetylgalactosamine

GalNAc

N-acetylglucosamine / N-acetylglucosamine-transferase

GlcNAc / GlcNAc-T

N-acetylneuramic acid

Neu5Ac

normal phase

NP

neural cell adhesion molecule

NCAM

peptide:N-glycanase A/F

PNGase A / PNGase F

photoactivated localization microscopy

PALM

polyethylene tetraphthalate

PET

polysialic acid

PolySia

polyvinylidene fluoride

PVDF

polyvinylpyrrolidone 40

PVP40

porous graphitized carbon

PGC

proline

Pro

pyrophosphate

PP

reversed-phase

RP

ribonuclease

RNase

room temperature

RT

rough endoplasmic reticulum

rER

serine

Ser

sialic acid / sialyl-transferase

SiaAc / Sia-T

sialyl Lewis X

SLeX

sodium cyanoborohydride

NaBH3CN

sodium hydroxide

NaOH

solid phase extraction

SPE

tandem mass spectrometry

MSn

threonine

Thr

transforming growth factor β

TGF-β

trifluoroacetic acid

TFA

tryptophan

Trp

tyrosine

Tyr

ultraviolet

UV

uridine-5’-diphosphae

UDP

zwitterionic

ZIC

Abbreviations were considered in the List of Abbreviation, if they appear at least two times in the main text.

XI

Introduction

1

Introduction

1.1

Glycobiology

Carbohydrates are usually encountered in the context of energy metabolism, e. g., breakdown of glucose (Glc) or glycogen. Although glycogen is linked to a protein core, the function of sugar molecules as storage form and its breakdown in energy metabolism falls outside the field of glycobiology. This distinction allows to focus on other functions of sugars, which are less well understood (Taylor and Drickamer 2011) and common among non-glycobiologists. Carbohydrates have far more important biological roles in the assembly of complex single- and multicellular organisms. All cells and numerous macromolecules carry an array of attached complex carbohydrate chains which are referred to as glycans. (Varki et al. 2008). Glycans encompass free or unconjugated complex oligo- and polysaccharides (Cummings and Pierce 2014) or attached mono-, oligo-, and polysaccharides. Their most common constituents are hexoses but important exceptions such as sialic acids (SiaAc) exist. SiaAc are a large family of nine carbon sugars (Alley and Novotny 2010, Taylor and Drickamer 2011) bearing a negative charge at physiological conditions (Dall'Olio and Chiricolo 2001). Until 2009, about 50 SiaAc have been described (Schauer 2009, Galuska et al. 2010). N-glycolylneuramic acid (Neu5Gc) and N-acetylneuramic acid (Neu5Ac) are the most prevalent sialic acid in mammalian cells. The latter appears in human mammalian cells (Zhou et al. 2017). The ring of Sia Ac is formed by six of its nine carbons as shown in Fig 6 (Jäckh 1976). Glycans attached to macromolecules are referred to as glycoconjugates and appear as glycoproteins, proteoglycans, glycolipids, and glycosylphosphatidylinositol (GPI) anchor (Roseman 2001, Taylor and Drickamer 2011, Jensen et al. 2012). GPI describes a unique form of glycosylation, in which the protein is linked to a lipid anchor through a glycan chain. Due to numerous glycan acceptors on the membrane, all cell surfaces are coated with a thick layer of glycans (Zaia 2008, Cummings and Pierce 2014) and serves first level of interaction with its environment (Jensen et al. 2012). This specific layer of each cell is called glycocalyx (Lindhorst 2000). The glycocalyx is shown in electron microscopy in Fig 1. Over 10 million glycans on the cell surface are provided through N- and O-linked glycoproteins (Cummings and Pierce 2014).

Fig 1: Glycocalyx of an Erythrocyte. Electron microscopy picture of stained glycans. The glycocalyx on the cell surface is up to 1,400 Å thick, and glycan filaments are 12 – 25 Å in diameter. Source: Roseman (2001), page 41528.

1

Introduction

Another term used in the field of glycobiology is glycomics which is defined by the study of all glycoconjugates and glycans expressed within a biological system (Zaia 2008). The mammalian glycome repertory encompasses thousands of glycan structures, possibly even larger than the proteome (Ohtsubo and Marth 2006). The study of glycomics is also dependent on other “−omic” studies as their acceptors, metabolites, and products influence the biosynthesis of glycans (Cummings 2009, Hart and Copeland 2010) as shown in Fig 2.

Fig 2: Association of Glycomics and Other “-Omics” Studies. Glycan structures are regulated by monosaccharide metabolism (metabolomics), glyco-enzyme expression (genomics and transcriptomics), and glycan acceptors (proteomics and lipidomics). Bioinformatic tools are used for structure analysis. Detailed knowledge of all “-omic” fields is indispensable to identify biomarkers and developing of drugs. Abbreviations: CDP = cytidine 5’-diphosphte; GDP = guanosine 5’-diphosphate; UDP = uridine 5’diphosphate Source: Hart and Copeland (2010), page 675.

In summary, the field of glycobiology is focused upon understanding the biosynthesis, chemistry, structures, and biological functions of glycans (Ohtsubo and Marth 2006). Glycans are secondary gene products (Fig 3) that cannot be predicted from analysis of genomic deoxyribonucleic acid (DNA) sequences (Ohtsubo and Marth 2006). Carbohydrate composition on the cell surface is influenced by their specific intracellular glycosylation machinery. Oligosaccharide composition varies from cell type to cell type. Information gained from different glycoprofiles can be used to discover changes in glycosylation and to identify biomarkers that occur in cell differentiation and diseases (Jensen et al. 2012). In literature, glycans are often described as oligosaccharides (Taylor and Drickamer 2011). Although this designation may be correct in many ways, some glycans include more than ten sugar components. Therefore, this designation is here considered as insufficient and not used as synonym for glycans in this work.

2

Introduction

Fig 3: Interaction of Glycoconjugates with Cellular Processes. Glycans are secondary gene products that mediate cell-cell as well as cell-matrix interactions. Mediating glycan structures through enzymatic reactions are under complex regulation. Abbreviations: DNA = deoxyribonucleic acid; ECM = extracellular matrix; NCP = non-collagenous proteins; RNA = ribonucleic acid. Source: Zaia (2008), page 882.

1.2

General Biological Function of Glycans

Glycans exert their biological functions through molecular mechanisms involving direct glycan recognition as well as indirect glycan contribution to expression and conformation of glycoconjugates (Cummings and Pierce 2014). Cell surface glycans are directly recognized by glycan-binding proteins (GBPs), and therefore promote molecular interactions like cell adhesion of immune cells (Sperandio et al. 2009, Gu et al. 2012, Cummings and Pierce 2014, Yang et al. 2015, Zhou et al. 2017). But intra- and intermolecular binding can also be inhibited by glycans (Ohtsubo and Marth 2006). Furthermore, glycans play vital roles in many important cellular events such as protein halflife, protein folding, immune cell adhesion, bacterial adhesion, cell-cell interaction, modulation of endocytosis and signal transduction (Rodan et al. 1996, Ohtsubo and Marth 2006, Wuhrer et al. 2009b, Kamiya et al. 2012, Reiding et al. 2014, Kolarich et al. 2015). Glycans influence signal transduction through interactions of ligands and receptors with themselves as well as distinct membrane domains (Fig 4), and by co-regulation of molecules (Ohtsubo and Marth 2006). Endocytosis of the epidermal growth factor (EGF) and transforming growth factor β (TGF-β) receptor can be regulated by glycans containing N-acetylglucosamine (GlcNAc) in α1,6position, which is catalyzed by GlcNAc-transferase (T)-V. Receptor endocytosis of EGF and TGF-β will be retarded, especially in carcinoma cells, leading to alterations in receptor activation and signal transduction (Ohtsubo and Marth 2006). Glycans play also a significant role in the immune system. For example, glycans at the surface of pathogens are recognized by lectins (Ohtsubo and Marth 2006) resulting in activation of the cellular and humoral immune system (Wuhrer et al. 2009b). Glycoconjugates in sweat do also contribute in microbial adhesion as first line of defense (Peterson et al. 2016). On the other hand, defects in glycosylation can influence the self-/non-self-recognition leading to numerous autoimmune diseases at worst (Ohtsubo and Marth 2006). 3

Introduction

Those described mechanisms are accomplished through direct glycan recognition. Glycans can also exert glycoprotein stability, conformation, oligomerization, turnover and cell surface resident time through indirect mechanisms (Cummings and Pierce 2014). The involvement of glycans in glycoprotein function, direct glycan recognition and indirect glycan effects are shown in Fig 4.

Fig 4: Main Roles of Glycans in Glycoprotein Functions. Glycans in glycoproteins are involved in recognition by GBPs, and indirect glycan effects on glycoprotein interactions, depending on protein-protein, lipid-protein, and glycan-glycan interactions. Abbreviations: GBP = glycan-binding protein; O-GlcNAc = O-β-N-acetylglucosamine. Source: Cummings and Pierce (2014), page 2.

1.2.1 Glycosidic Bonds and Linkages Unlike proteins, which have limited numbers of diverse peptide bounds, glycosidic bondages and therefore their structures express a great versatility. Since monosaccharides bear multiple hydroxyl moieties, many different O-glycosidic bonds are possible as shown in Fig 5. Furthermore, glycosidic linkages can be distinguished between α and β depending on stereochemistry relation of the exocyclic oxygen to its stereodefined carbon atom. The αglycosidic bonds are in trans-, while β-glycosidic bonds are in cis-configuration (Lindhorst 2000, Berg et al. 2012, Hua et al. 2012). Regarding all feasible glycosidic linkages between an α-anomeric C1 of one sugar to all other hydroxyl moieties of another monosaccharide, isoforms of each monosaccharide, and possible glycosidic linkages (α/α, α/β, β/a, and β/β) reveal the possibility of inconceivable structural diversity. In fact, three different monosaccharides can be linked together in thousands of different structures. Due to their structural diversity glycans may exert various and/or similar functions (Lindhorst 2000, Berg et al. 2012, Hua et al. 2012) as described in Section 1.2. 4

Introduction

Fig 5: Brief Summary of Glycosidic Bondages and Branching Points. A shows two Glc molecules linked by α1,4-glycosidic bond to form maltose; B represents an integral part of glycogen with additional α1,6-linkages leading to branching points; C and D show distinct structural outcomes by repetitive homogenous α1,4 and α1,6 linkages of Glc. Note: A and B are shown in Haworth, while C and D are displayed in chair form. Abbreviations: Glc = glucose. Source: Berg et al. (2012), pages 327 – 329.

1.2.2 Monosaccharide Components of Mammalian Glycans Mammalian glycans are built from nine monosaccharides (Cummings and Pierce 2014) which are provided through diet (Ohtsubo and Marth 2006) or endogenous synthesis. The common monosaccharides are Glc, GlcNAc, galactose (Gal), N-acetylgalactosamine (GalNAc), mannose (Man), fucose (Fuc), glucuronic acid (GlcA), xylose (Xyl) and Neu5Ac. A tenth sugar, iduronic acid (IdoA) is created within presynthesized glycosaminoglycans (GAGs) (Moremen et al. 2012, Cummings and Pierce 2014). The monosaccharide Neu5Ac is shortly introduced due to its specific nature. As noted in Section 1.1, Neu5Ac is the predominantly SiaAc in mammalian cells. Neu5Ac is widely distributed in both, N- and O-glycans, respectively (Zhou et al. 2017). Terminate Neu5Ac binds human lectins (Macauley et al. 2014) and acts also as anti-recognition agent (Schauer 2009). It is furthermore important for cellular communication and determines protein half-life (Reiding et al. 2014). SiaAc are mostly attached to terminal Gal through α2,3- or α2,6-glycosidic linkage as shown in Fig 6. Moreover, Neu5Ac appears in polysialic acid (PolySia) as unique linear homopolymer with α2,8- and α2,9-glycosidic linkages (Nakata and Troy 2005). PolySia is predominantly found on tumors or neural tissues where it is linked to glycans of the neural cell adhesion molecule (NCAM). NCAM influences neural functions and plasticity (Galuska et al. 2007) and due to its large negative charge it inhibits homotypic NCAM binding (Ohtsubo and Marth 2006). Transfer of Neu5Ac appears linkage and 5

Introduction

substrate specific by a family of 20 Sia-T (Dall'Olio and Chiricolo 2001, Crocker et al. 2007, Schauer 2009). The linkages have indeed various functional consequences. For example, α2,3-linked Neu5Ac are required for sialyl Lewis X (SLeX) structures, while α2,6-linked Neu5Ac facilitate cell survival via galectin inhibition (Reiding et al. 2014).

Fig 6: Two Most Common Neu5Ac Linkages in Mammals. Abbreviations: Neu5Ac = N-acetylneuramic acid; Gal = galactose. Source: Crocker et al. (2007), page 259.

1.2.3 Glycans in Health and Disease Glycan processing is highly sensitive to alterations in intracellular biological mechanisms which can alter to different physiological states, like aging, as well as to pathophysiological conditions (Rudd et al. 2001, Alvarez-Manilla et al. 2007, Aoki et al. 2007, Stumpo and Reinhold 2010, Jensen et al. 2012, Miura and Endo 2016). The enlightenment of those changes contributes potential for diagnosis and prognosis of diseases (Sethi et al. 2016, Zhou et al. 2017) and could even advance personalized medicine (Hinneburg et al. 2017). Monoclonal antibodies expressed from mammalian cell lines in vitro are of intense focus for the pharmaceutical industry (Prater et al. 2009, Benet and Austin 2011). The anti-thrombotic glycan heparin, for example, is approximately a billion times prescribed annually (Ohtsubo and Marth 2006). Before pharmacological approaches are even conceivable, glycoproteins have to be characterized first, including identification of glycoproteins of interest, determination of glycosylation site, and characterization of glycan structures, including linkages, branch points and their relative abundances (Mysling et al. 2010). Unfortunately, this work cannot emphasize and characterize the vast amount of diseases associated with changes in glycosylation. A brief insight is given in the following paragraphs. Currently, many research groups have made a lot of effort to study changes on cell surface glycosylation on tumor tissues (Ishimura et al. 2006, Lau and Dennis 2008, Lattova et al. 2009, Wuhrer et al. 2009b, Miwa et al. 2012, Reiding et al. 2014, Ruhaak et al. 2015). One example is the formation of SLeX structures, including α2,3-linked Neu5Ac, which are considered as possible indication of metastasis (Fig 7) for several cancer types (Matsuda et al. 2008, Reiding et al. 2014). Progress has also been done in studying glycosylation patterns in rheumatoid arthritis. Levels of fully galactosylated sugars decrease in the course of the disease and reflect, therefore, a decrease Gal-T activity (Rudd et al. 2001). Inherited errors of glycosyl-transferases or glycosidases, responsible for proper glycosylation are referred to as congenital disorder of glycosylation (CDG). Their consequences may lead to serious malfunction of several organ systems (Guillard et al. 2011, Reynders et al. 2011, 6

Introduction

Berg et al. 2012). CDGs are subdivided into two groups: Type I defines mutated genes affecting the addition of N-glycans while type II encompasses their processing (Freeze 2013). Deficiency of GlcNAc-T I lead to severe embryonic defects and morphogenic abnormalities like aberrant vascularization or defects with situs inversus of heart loop formation. Interestingly, this deficiency is well tolerated among different cell lines (Ohtsubo and Marth 2006), but not in vivo. Another example is the absence of α-mannosidase II and α-glycosidase which are necessary in non-immune cells for regular N-glycan synthesis. Their deficiency can induce the systemic autoimmune disease lupus erythematosus (Ohtsubo and Marth 2006) leading to hyperactive immune system attacking healthy tissues (Morel 2017). Defects in degrading proteins and glycans due to glycosidase deficiencies leads to cellular storage disorders such as Niemann-Pick type C, Gaucher’s disease, Tay-Sachs, and Sandhoff’s disease. Fortunately, these disease can clinically be treated through an organic compound that inhibits Glc-T I activity (Ohtsubo and Marth 2006). More insights to CDGs are published in Freeze (2013).

Fig 7: Changes in Protein Glycosylation during Cellular Transformation and Progression. Changes in protein glycosylation on soluble glycoproteins and on the membrane are typical during early and/or late cancer progression. Changes are shown in pink-boxed areas. Abbreviations: Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; Man = mannose; Neu5Ac = N-acetylneuramic acid; Neu5Gc = N-glycolylneuramic acid; SLeX = sialyl Lewis X. Source: Stowell et al. (2015), page 481.

1.2.4 Glycans in Clinical Use Progress has been made in the developing therapeutic glycoproteins in clinical use. For instance, human tissue-type plasminogen activator (tPA) is used as therapeutic glycoprotein which functions in dissolving blood clots preventing atherosclerosis and stroke. The glycoprotein erythropoietin (EPO), which stimulates the production of erythrocytes, is applied to patients with renal failure or cancer. Interestingly, non-glycosylated EPO was more active than its N-glycosylated counterpart in in vitro studies, however, N-glycosylated EPO has a

7

Introduction

higher efficacy in vivo. More precisely, the more N-glycan sites the higher circulatory half-life and efficacy as drug (Jones et al. 2005). Unfortunately, dietary intake of specific glycans or monosaccharides has not been considered beneficial in disease treatment or on human health, except in cases of rare genetic diseases (Ohtsubo and Marth 2006). But exercise seems to have a positive influence on the hexosamine biosynthetic pathway (Shirato et al. 2012) and O-GlcNAc modification (Cox and Marsh 2013).

1.3

Protein Glycosylation

Glycosylation of proteins is one of the most common co- and posttranslational modifications (Mamedov and Yusibov 2011, Burnina et al. 2013) occurring in over 50% of all gene products (Balaguer and Neususs 2006, Zaia 2008, Aldredge et al. 2012, Nagae and Yamaguchi 2012). In vivo biological functions of glycoproteins are influenced by their specific glycosylation (Prater et al. 2009, Kolarich et al. 2015) at one or more amino acids (AA). Almost all nuclear and DNA binding proteins, cytoplasmic enzymes, secreted proteins, membrane proteins and some mitochondrial proteins are glycosylated (Jones et al. 2005, Cummings and Pierce 2014). As mentioned in Section 1.2, glycans have a protective influence on protein half-life through steric hindrance to proteases. The absence of carbohydrate chains may cause random or nonspecific proteolysis (Bernard et al. 1983). Most glycoproteins have several glycoforms (Jensen et al. 2012). Glycoforms are the same gene product with (slightly) different glycans at their glycosylation site (Kuster et al. 1997, Desantos-Garcia et al. 2011, Taylor and Drickamer 2011). Up to 100 alternative glycans at a single glycosylation site may occur. Some glycans confer specific features on their protein by acting as recognition epitope (Rudd et al. 2001). Two identical enzyme bearing different glycans catalyze the same reaction, but the function of its glycan may differ (Taylor and Drickamer 2011) and/or affect solubility, immunogenicity, stability, degradation, efficacy and activity (Jensen et al. 2012). On the other hand, two completely different enzymes may bear the same or similar glycan (Taylor and Drickamer 2011), and their clearance may be initiated through common receptors (Fig 8).

Fig 8: Glycoforms. The polypeptide and glycan portion of glycoproteins may have potential independent functions. Different glycoproteins may bear identical or similar glycans and different gene products can be glycosylated heterogeneously. Source: Taylor and Drickamer (2011), page 14.

8

Introduction

A total of nine AAs can be glycosylated in nature, including asparagine (Asn), serine (Ser), threonine (Thr), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), hydroxylysine (Hyl), and hydroxyproline (Hyp) (Cummings and Pierce 2014). Proteoglycans are a subclass of glycoproteins, consisting of a core protein which is covalently attached to at least one GAG. In contrast to glycoproteins, proteoglycans are far more heavily glycosylated (Ohtsubo and Marth 2006). The GAG makes up as much as 95% of the molecular weight. Thus, proteoglycans resemble more a polysaccharide than a protein (Berg et al. 2012). Glycans attached to glycoproteins are subdivided into two major classes. Glycans, attached to polypeptide structures via amide linkage to Asn side chains are defined as N-glycans. Glycosidic linkages to side chains of Ser/Thr, Tyr (glycogenin), and Hyl (collagen) are referred to as O-glycans (Moremen et al. 2012). N- and mucin-type O-glycans are, until now, the best studied forms (Hinneburg et al. 2017). Beside nitrogen and oxygen linkages, less common linkages also occur. For instance, carbon linkages to the C2 position of Trp are considered as C-mannosylation (Moremen et al. 2012). Tab 21 (Appendix) gives a short overview of mentioned glycosylation types. An overview of major classes of mammalian glycan structures are given in Fig 9. In contrast, glycation is a non-enzymatic and irreversible process that is common in various diseases (Ohtsubo and Marth 2006), like diabetes, and may also be a factor in aging (Miura and Endo 2016). Glycans are synthesized through a portfolio of cellular enzymes and substrates. Approximately 1 – 2% of the genome encompasses information for the glycan machinery (Fig 3). Approximately 200 glycosyl-transferases extend acceptor glycans using lipid-linked or nucleotide sugars as activated donor substrates (Moremen et al. 2012). Their specification is mostly determined by glycan structure instead of specific peptides sequences (Ohtsubo and Marth 2006). Glycosyl-transferases with overlapping glycan acceptors but different donors do compete to each other leading to diverse glycan structures (Moremen et al. 2012). Moreover, glycosidases responsible for hydrolyzation of specific glycosidic linkages collaborate as well in glycan biosynthesis. Gene transcription of both, glycosyl-transferases and glycosidases, is regulated at posttranscriptional and posttranslational level, resulting in isoenzymes and therefore, diverse glycan structures. Glycosyltransferases and glycosidases may also be regulated by phosphorylation (Ohtsubo and Marth 2006). Furthermore, changes in the glycome can be induced by loss of chaperones or multiprotein complexes, which have an impact on glycosyl-transferases trafficking between the rough endoplasmic reticulum (rER) and Golgi apparatus. (Ohtsubo and Marth 2006). Acetyl-transferases and sulfo-transferases are technically no part of the glycosylation machinery, although they attach functional acetyl and sulfate groups to carbohydrates residing on glycans. Subsequently, glycan structure and function are modulated (Ohtsubo and Marth 2006).

9

Introduction

Fig 9: Major Classes of Mammalian Glycan Structures. This figure gives a pictorial overview of glycoproteins and proteoglycans. Most glycans on secreted and membrane proteins are found in N- or O-linkages. N-linked glycans can occur in high-mannose, complex and hybrid structure. Other glycans shown in this figure are also listed in Tab 21 with their corresponding sequence motif. Source: Moremen et al. (2012), page 450.

1.3.1 N-Glycans N-glycosylation occurs usually at the Asn residues in the sequence Asn-X-Ser/Thr while X can be any AA except of proline (Pro) (Yamaguchi and Uchida 1996, Ruddock and Molinari 2006, Banerjee 2012) as it would inhibit the core glycosylation (Jones et al. 2005). Asn-X-Thr is more efficiently glycosylated than Asn-X-Ser (Jones et al. 2005). Less than 1% of N-glycans have the motif Asn-X-Cys or Asn-glycine (Gly)-Gly-Thr, respectively (Jones et al. 2005, Cummings and Pierce 2014). The AA in position Y (Asn-X-Ser/Thr-Y) has also an influence on the efficiency of core glycosylation. Cys in position Y has an inhibitory effect through possible formations of disulfide bonds (Jones et al. 2005). Unlike other post-translational modifications such as phosphorylation or even O-glycosylation, no further protein domain is necessary to define the N-linked glycosylation site (Aebi et al. 2010). All N-glycans share a common core sugar sequence GlcNAc2Man3 (Zaia 2008) and are classified into three types. The core of high-mannose or oligomannose, respectively, is only 10

Introduction

attached to Man residues. In complex-type glycans are at least one antennae attached to the core initiated by GlcNAc-T. Hybrid-type glycans are a mixed type of both. An only Man residue is attached to the α1,6-Man arm, and one or two antennae are attached to the α1,3-Man (Varki et al. 2008). Types of N-glycans are shown in Fig 10. N-glycans influence many properties of glycoproteins as mentioned in Sections 1.2, and 1.3, including protein stability, solubility, antigenicity, half-life, clearance rate, and in vivo activity (Jones et al. 2005). Other major roles of N-glycans have been supposed to be the intracellular trafficking of glycoproteins (Cummings and Pierce 2014) as well as facilitation of nascent polypeptide folding (Grafl et al. 1987). The renaturation of reductively denatured pancreatic ribonuclease (RNase) B can also be promoted by high-mannose type glycans (Marquardt and Helenius 1992) through depressing unfavorable intramolecular interactions of the polypeptides (Nishimura et al. 1998). Due to their extensive branching and larger size N-glycans are the second most complicated structures to analyze right after GAG. For instance, tetra-antennary N-glycans may consist of heterogeneous branching linkages which are even isobaric (Cummings and Pierce 2014). The analysis of such structures does indeed require solid training (Taylor and Drickamer 2011).

Fig 10: Types of N-Glycans Added to Polypeptides at Asn-X-Ser/Thr Sequons. Abbreviations: Asn = asparagine; Ser = serine; Thr = threonine; X = any amino acid except of proline. Source: Varki et al. (2008), chapter 8.

1.3.2 O-Glycans O-glycans are generated by elaboration of GalNAc (Zaia 2008) to the hydroxyl group of Ser or Thr (Rudd et al. 1997) with far more unanimous motifs as N-glycans (Tab 21). An O-linked monosaccharide, GlcNAc-β-Ser/Thr, is also found in numerous cytoplasmic and nuclear proteins. This glycosylation has shown to be competing with Ser/Thr phosphorylation (Moremen et al. 2012). O-linked glycans are highly diverse in structure as well as in function, but their full extent of their diversity has not been established yet (Taylor et al. 1996). Unlike N-glycan synthesis which occurs in the rER and Golgi apparatus, initiation of O-glycosylation starts in the Golgi (Zhang and Chelius 2004). Some structural and motif information are given in Fig 4, Fig 9, and Tab 21.

11

Introduction

1.4

Synthesis of N-Glycans

N-glycan synthesis starts on the cytoplasmic side of the rER. It begins with GlcNAc that is linked through a high-energy bond to a nucleotide-activated sugar donor, uridine 5’diphosphae (UDP), forming UDP-GlcNAc (Herscovics 1999, Munro 2001). The membraneassociated

GlcNAc-1-phosphotransferase

(Jones

et

al.

2005),

transfers

GlcNAc-

phosphate (P) to dolichol-P (Dol-P) which serves as a lipid anchor for the assembly of a glycan (Aebi et al. 2010). Dol is a hydrophobic lipid, consisting of multiple isoprene units (15 – 19), such as those in cholesterol, but not cyclized (Jones et al. 2005, Taylor and Drickamer 2011). Its structure is shown in Fig 11.

Fig 11: Structure of Dolichol Phosphate. Source: Berg et al. (2012), page 334.

Dol is synthesized in the cholesterol biosynthesis pathway through diverging reactions of isopentenyl PP or farnesyl PP (Jones et al. 2005). It plays a major part in decreasing the fluidity and disrupting the membrane bilayer at high local concentrations (Jones et al. 2005).

1.4.1 Synthesis of N-Glycan Precursor in the Rough Endoplasmic Reticulum Back to the N-glycan precursor synthesis, another GlcNAc and five Man residues are transferred from UDP-GlcNAc and guanosine 5’-diphosphate (GDP)-Man in a sequential manner, creating the intermediate Dol-PP-GlcNAc2Man5. It is assumed that the bilayer instability due to rising [Dol-PP-glycan] serves to assist the “flipping” of the glycan intermediate to the luminal side of the rER (Jones et al. 2005, Samuelson and Robbins 2015) as shown in Fig 12. Once inside the rER, four more Man and three more Glc residues are further attached through luminal glycosyl-transferase, using Dol-P-Man/Glc as substrates to complete the lipidlinked oligosaccharide (LLO) moiety (Moremen et al. 2012), Dol-PP-GlcNAc2Man9Glc3 which is shown in Fig 13. The next step involves the en bloc transfer of GlcNAc2Man9Glc3 onto potential glycosylation sites, e. g., Asn-X-Ser/Thr, of nascent polypeptides. This reaction is mediated by an oligosaccharyl-transferase (Jones et al. 2005). The overall number of N-glycan attachment is increased by performing glycosylation during or instantly after translocation of the nascent protein into the rER (Aebi et al. 2010). The PP remains at Dol (Fig 12) which will be dephosphorylated and consequently flip back to the cytoplasmic site (Jones et al. 2005) ready for the next GlcNAc. The glycan precursor, GlcNAc2Man9Glc3 (Fig 13), has a half-life of a few seconds (Aebi et al. 2010). Once it is transferred to the nascent protein, the glycan undergoes trimming and processing immediately (Jones et al. 2005). It begins with the outermost Glc residue (Fig 13n) by a membrane bound α1,2-exoglucosidase, glucosidase I (Aebi et al. 2010, Roth and Zuber 2017). 12

Introduction

Fig 12: Topological Model of N-Glycan Synthesis in the rER. The first GlcNAc is transferred from UDP-GlcNAc to Dol-P, leading to Dol-PP. Further sugar donors synthesized in the cytoplasm are transferred to the LLO in a stepwise manner to form Dol-PP-Glc5Man5. This intermediate flip into the lumen of the rER where further Man and Glc donors are attached until Dol-PP-GlcNAc2Man9Glc3 is formed. This glycan is then transferred en bloc to a nascent protein. Nucleotide linked Man and Glc are synthesized in the cytoplasm and translocated via Dol-P into the lumen of the ER. Colors and shapes do not comply with structure motifs of CFG. Abbreviations: CFG = Consortium for Functional Glycomics; Dol = dolichol; GDP = guanosine 5’diphosphate; Glc = glucose; GlcNAc = N-acetylglucosamine; LLO = lipid-linked oligosaccharide; Man = mannose; OST = oligosaccharide transferase; P = phosphate; PP = pyrophosphate; rER = rough endoplasmic reticulum; UDP = uridine 5’-diphosphae. Source: Jones et al. (2005), page 123.

The remaining GlcNAc2Man9Glc2 is further processes through an α1,3-exoglucosidase which removes the next Glc residue (Fig 13m). The generated GlcNAc2Man9Glc function as substrate for rER-resident lectin chaperones calnexin (transmembrane protein) and calreticulin (soluble homolog of calnexin). One domain acts as carbohydrate-binding domain and the other recruits the protein disulfide isomerase A3 (PDIA3), also referred as glucose-regulated protein 58kD (GRP58) (Aebi et al. 2010). This enzyme catalyzes the formation of native disulfide bonds in folding polypeptides. Furthermore, it prevents nascent polypeptides from non-productive disulphide bonding as well as from hydrophobic aggregation (Moremen et al. 2012). Upon release of the folding polypeptide from the calnexin/calreticulin cycle, glucosidase II removes the innermost glucose (Fig 13l) remaining GlcNAc2Man9 (Aebi et al. 2010, Roth and Zuber 2017). Persistently misfolded glycoproteins lose a single Man residue through hydrolysis by mannosidase I, leading to retro-translocation to the cytoplasm where glycoproteins are deglycosylated, ubiquitinated and degraded in the proteasome. This process is known as ER associated degradation (ERAD) (Jones et al. 2005, Roth and Zuber 2017). Appropriate folded glycoproteins are further trimmed in the lumen of the rER. The α-mannosidase I hydrolysis the terminated Man residue (Fig 13g). N-glycan synthesis in the rER usually ends at this point and continues in the Golgi-apparatus.

13

Introduction

Fig 13: Glycan Precursor attached to LLO or N-Glycosylation Site. Abbreviations: LLO = lipid linked oligosaccharide; N = asparagine; S = serine; T = threonine; X = any amino acid except of proline. Source: Aebi et al. (2010), page 75.

1.4.2 N-Glycan Synthesis in the Golgi-Apparatus The glycoprotein with its attached GlcNAc2Man8 is translocated to the cis-Golgi where the processing continues (Jones et al. 2005, Moremen et al. 2012) in nontemplate-controlled manner (Zaia 2008). However, some glycans remain in this state or run only through minor changes. These glycans are called high mannose or oligomannose N-glycans (Fig 10). Other glycans are further processed. Complex glycans are stripped of Man through different specific mannosidases until five Man are left (GlcNAc2Man5). The removed Mans are shown in Fig 13f, Fig 13i, and Fig 13k. The procedure continues with re-elongation by GlcNAc-T I which is located in the medial-Golgi. GlcNAc-T I attaches a β1,2-linked GlcNAc to the Man shown in position Fig 13d. Next, two additional Man residues (Fig 13h and Fig 13j) are removed through mannosidase II, followed by further attachment of GlcNAc in β1,2-linkage to Man (Fig 13e) by GlcNAc-T II. This result in bi-antennary complex N-glycan as it is shown in Fig 10. Tri-, tetra-, and penta-antennary complex N-glycans are also common. For their synthesis, additional bisecting GlcNAc-Ts are required. GlcNAc-T III adds a β1,4-linked GlcNAc to Man in position Fig 13c, GlcNAc-T IV attaches a β1,4-linked GlcNAc to Man in position Fig 13d, and GlcNAc-T V adds a β1,6-linkied GlcNAc to Man in position Fig 13e (Taylor and Drickamer 2011). Further GlcNAc-T and other linkages are also described in the literature (Varki et al. 2008). Fuc-T are also present in the medial-Golgi and may attach α1,6-linkaged Fuc to GlcNAc in position Fig 13a. In the trans-Golgi, Gal-T (e. g., β1,4-linkages), Fuc-T (e. g., α1,3- and α1,6-linkages), and SiaT (e. g., α2,3- and α2,6-linkages) add further monosaccharide donors (Taylor and Drickamer 2011). Neu5Ac decorates glycans usually at the distal portion (Dall'Olio and Chiricolo 2001). In any case, other glycosyl-transerases are present in the trans-Golgi, attaching other linkages 14

Introduction

or monosaccharides. The vast amount of glycosyl-transferases and glycosidases, as mentioned in Section 1.3, cannot be discussed in more detail at this point. An overview of glycan processing across the Golgi apparatus is shown in Fig 14.

Fig 14: Glycan Processing in the Golgi Apparatus. Abbreviations: CDP = cytidine 5’-diphosphte; CMP = cytidine 5’-monophosphate; ER = endoplasmic reticulum; Fuc = fucose; Gal = galactose; GDP = guanosine 5’-diphosphate; GMP = guanosine 5’monophosphate; GlcNAc = N-acetylglucosamine; Man = mannose; UDP = uridine-5’-diphosphate; UMP = uridine-5’-monophosphate. Source: Moremen et al. (2012), page 454.

15

Methodological Principles

2

Methodological Principles

2.1

Aim and Hypothesis of this Work

The first chapter, especially Sections 1.2 and 1.3, clarified that detailed knowledge on the particular structures associated with different cell types are crucial to understanding the functional roles of N-glycans in health and disease (Hinneburg et al. 2017). Monitoring protein glycosylation has been on focus since decades (Yuen et al. 2002). However, current state of the art methods cannot exactly assign glycosylation patterns of heterogenous tissues to specific cells. Cell specific analysis of glycosylation is indeed possible with cell lines in vitro. But their results do not always match with outcomes under in vivo conditions as it was described previously in Section 1.2.3. The glycocalyx or glycome do even change under regular physiological conditions, e. g., during spermatozoa differentiation (Brohi and Huo 2017) or mating seasons in animals (Accogli et al. 2017). Therefore, analysis of glycosylation in specific cellular states is needed to gain detailed insight into the role of glycans under different physiological as well as pathophysiological conditions (Rudd et al. 2001, Alvarez-Manilla et al. 2007, Aoki et al. 2007, Stumpo and Reinhold 2010, Jensen et al. 2012, Miura and Endo 2016). This goal may be achieved by using formalin-fixed paraffin embedded (FFPE) tissues (Section 2.3) which are available in many laboratories over the world (Casadonte and Caprioli 2011). Tissue sections comprise of various cells (Lehmann et al. 2000, Liotta and Petricoin 2000, Liu 2010, Vandewoestyne et al. 2012, Hinneburg et al. 2017). For example, tumor tissues are surrounded by inflammatory cells, stromal cells and migratory cells (Cheng et al. 2013). The analysis of whole FFPE tissue sections would not deliver valuable information about the connection between each glycan to its cell. To overcome this issue laser capture microdissection (LCM) should be considered to extract cells or areas of interest (Section 2.4). After releasing glycans of selected cells (Section 2.5), analysis continues through various bioanalytical instruments (Sections 2.6 and 2.8). Bartel et al. (2014) used this LCM successfully to analyze SiaAc of selected cells. This thesis continues this work. Starting from scratch, only N-glycans were considered in this work. Many different devices have been established for carbohydrate analysis and characterization. Common bioanalytical devices for N-glycan analysis used so far are lectin affinity chromatography, high performance liquid chromatography (HPLC), porous graphitized carbon (PGC) chromatography, high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography (GC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These techniques can be used complementary to each other in many ways, each with its own advantages and disadvantages (Fu et al. 1994, Morelle et al. 2005, Wuhrer et al. 2009b, Westphal et al. 2010, Kozak et al. 2015). Due to the complexity of glycan linkages and many possible isomers, a single-method bioanalytic technique is probably an unreasonable goal for glycan analysis (Harvey 1999).

16

Methodological Principles

The experiments started with the establishment of permethylation of (sialylated) N-glycans (Section 3.7) and their analysis (Section 3.8) using matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) MS (middle branch in Fig 15). The study continued with normal phase (NP)-HPLC using a hydrophilic interaction liquid chromatography (HILIC) column. The aims were establishing the needed amount of microdissected cells (Section 3.3), testing different antigen retrieval procedures (Section 3.4), optimizing the enzymatic N-glycan release (Sections 3.5.1 and 3.5.2), purification (Section 3.5.3), as well as derivatization (Section 3.5.4; right branch in Fig 15). Finally, it was planned to use a two-dimensional HPLC approach (Wuhrer et al. 2009b) and online MS analysis. Starting with NP-HPLC and continuing with assembled peaks in reverse phase (RP)-HPLC coupled to electrospray ionization (ESI)-MS (left branch in Fig 15). Once all bioanalytical data are obtained, glycan structures have to be interpreted preferably by bioinformatic tools (Maass et al. 2007) like GlycoWorkbench (Cummings and Pierce 2014). This approach can be considered as top-down N-glycan analysis. The overall goal of this thesis was the development of a protocol for N-glycan analysis of laser microdissected cells from FFPE tissue sections. It was hypothesized that laser capture microdissected FFPE tissue sections is a suitable approach to analyze N-glycans of specific cells or areas of interest. Korekane et al. (2007) and Hinneburg et al. (2017) have already used this approach for N-glycan analysis. Their results will be discussed in Section 5.5.

Fig 15: Methodological Concept of this Work. Abbreviations: Asn = asparagine; HILIC = hydrophilic interaction liquid chromatography; HPLC = high performance liquid chromatography; RP = reverse phase; Ser = serine; Thr = threonine Source: Adapted from Cummings and Pierce (2014), page 8.

17

Methodological Principles

2.2

Standards

2.2.1 Dextran The α-glucan dextran is composed of glucose chains of varying lengths. Its straight chain consists of α1,6-linkages while branches are linked by α1,3-bonds (Banerjee and Bandopadhyay 2016). Dextran was mainly used to adjust and verify the gradient elution mode of the HPLC system. N-glycans released from standard glycoproteins (Sections 2.2.2, 2.2.3, and 2.2.4) served as proof of principle.

2.2.2 Fetuin and Asialofetuin Bovine fetuin (fetuin B) has been used as a model for glycan and glycoprotein studies. It is structurally as well as biologically related to the human plasma α2 Heremans Schmid glycoprotein, also known as fetuin A. Its molar mass is 48 kDa. Fetuin B contains six glycans moieties of which each half are O-glycosidically and N-glycosidically attached. However, the N-glycans represent 80% of the glycans’ mass. The majority of Asn-linked oligosaccharides carry three peripheral branches and contain negatively charged moieties (Green et al. 1988). Various isomeric N-glycans are attached to fetuin B which are bi-, tri-, tetra-, and pentasialylated (Guttman et al. 1996). Tab 23 listed in the Appendix (Section 8.3) gives an overview of possible N-glycans attached to fetuin B. Asialofetuin is the desialylated version of fetuin and should differ from retention time and mass-to-charge ratio (m/z). Its use can be considered as first proof of principle when comparing to (sialylated) fetuin B.

2.2.3 Ribonuclease B Bovine pancreatic RNase B [EC 3.1.4.22] is a glycoprotein with a molecular mass of 15.5 kDa and contains one glycosylation site at Asn34 (Bernard et al. 1983). RNase B contains a single attached high Man type N-glycan with five to nine mannosyl residues (Fu et al. 1994). The most predominant sugar chain of RNase B is Man5GlcNAc2 (Berman et al. 1981, Prien et al. 2009). Unlike RNase B/C/D, RNase A is not glycosylated and often studied as counterpart to understand the role of high-mannose type N-glycans (Plummer and Hirs 1964). Known Nglycan structures are listed in the Appendix (Tab 24).

2.2.4 Human Transferrin Members of the transferrin superfamily are non-heme glycoproteins and responsible for irontransport. Their average molecular mass is between 74 – 82 kDa. Serum transferrin delivers iron to cells by a pH dependent, receptor-mediated process. Human serum transferrin has two N-glycosylation sites at Asn432 and Asn630. (Nagae et al. 2014). Isoforms of transferrin are expressed in other body compartments, for example cerebrospinal fluid (Hoffmann et al. 1995) and human semen. The latter is less prone to denaturation (D'Andrea et al. 1994). Enlightened N-glycan structures of human transferrin are represented in the Appendix (Tab 25).

18

Methodological Principles

2.3

Formalin-Fixed Paraffin Embedded Tissue

2.3.1 General information The majority of biopsies, e. g., tumor biopsies, are stored as FFPE tissues (Casadonte and Caprioli 2011) and used for histological analysis since over a century ago (Ly et al. 2016). FFPE tissues are of particular interest as they are storable for decades whereby they serve as pathology archives over the world (Hinneburg et al. 2017). The processing of FFPE tissues involves immersion in formalin for fixation by diffusion followed by dehydration in alcohol to remove fixative and residual water. The alcohol has to be removed from the sample by using xylene or a similar solvent, before the tissue is embedded in molten paraffin (Riedelsheimer et al. 2015, Ly et al. 2016). Fixation can be a critical step to ensure high-quality yields of (glyco)proteins. The quality of fixation is dependent on tissue size, temperature of fixation, and duration of fixative penetration (Espina et al. 2006). An initial rapid reaction of formaldehyde with basic AAs occurs when tissues are treated with formalin. This leads to formation of methylene bridges between amino residues, and therefore, formation of inter- and intramolecular cross-linked proteins (Metz et al. 2006). Thanks to the paraffin embedding (Ly et al. 2016) insoluble cross-linkages maintain histomorphological information over decades (Shi et al. 1991).

2.3.2 Deparaffinization Prior investigation of FFPE tissue sections wax-embedded specimen must be deparaffinized by using xylene (Casadonte and Caprioli 2011). Use of Roti®-Histol instead of xylene may be considered since it is less toxic. Roti®-Histol is manufactured from untreated orange peel and consist of 96 – 98% limonene (see manufacturers’ website). Deparaffinized sections should be washed in 100% alcohol, e. g., ethanol or isopropanol. Isopropanol, however, is cheaper and more mildly than ethanol. Staining of FFPE tissue sections allows morphological examination facilitating selection of various histological sections for bioanalytical approaches. Besides, staining enables the use of immunohistochemistry (IHC) techniques to label specific (glyco)proteins (Ly et al. 2016). If staining of histological sections is intended, samples shall be rehydrated by a graded alcohol series (Casadonte and Caprioli 2011). Sections are commonly stained with hematoxylin or Hemalaun in combination with eosin (Casadonte and Caprioli 2011, Gustafsson et al. 2012) also known as H&E. At last, tissue sections have to air-dry completely in order to prevent detachment of tissue section from histological glass slides (Casadonte and Caprioli 2011).

2.3.3 Antigen retrieval Beside all advantages of FFPE tissue sections formation of inter- and intramolecular methylene bridges leads to difficulties in analyzing of (glyco)proteins (Shi et al. 1991). Enzymatic N-glycan release is as well interrupted since N-glycans could be trapped within the cross-linked protein network (Ly et al. 2016). To overcome this problem, antigen retrieval is necessary to achieve partial reversal of cross-linkages (Shi et al. 1991). Antigen retrieval can 19

Methodological Principles

be accomplished by proteolytic digestion (Casadonte and Caprioli 2011, Ly et al. 2016) or a heat mediated process (Shi et al. 1991). The degree of cross-linkages may depend on the formalin fixation method (Taylor et al. 1996). Hence, optimal unmasking procedure needs to be tested for each tissue block (Casadonte and Caprioli 2011). Trypsin is a commonly used protease for antigen retrieval unless lysine (Lys) or Arg are preceded to Asn. In this case, another protease may be considered. Other potential proteases are chymotrypsin, pepsin (Kolarich et al. 2015), Pronase® (Hua et al. 2012), and proteinase K (Stavenhagen et al. 2017). Heating methods near 100°C can be advantageous as proteins will be denatured to a maximum extent (Casadonte and Caprioli 2011). In contrast, labile sugar molecules like SiaAc of sialylated glycans may be hydrolyzed through heat treatment (Karkas and Chargaff 1964, Varki and Diaz 1984, Thaysen-Andersen et al. 2013).

2.4

Laser Capture Microdissection

FFPE tissue sections are usually a mixture of heterogenous cellular subpopulation (Espina et al. 2006). Analysis of cell specific glycosylation patterns (Lindhorst 2000) indicates the need of microdissection (DeCarlo et al. 2011). Even various laser microdissection instruments are available today, “laser capture microdissection” or “LCM” is the generally used terminology regardless of laser type and method (Espina et al. 2006). Same applies to this work.

2.4.1 History The need for cell isolation has been debated for decades (Espina et al. 2007). Lowry and Passonneau (1972) pioneered the first freehand microdissection of specific cells under a microscope using razor blades, needles or fine glass pipettes back in 1972 (Hernandez and Lloreta 2006, DeCarlo et al. 2011, Cheng et al. 2013). Several publications followed describing freehand microdissection. Nevertheless, those techniques had too much shortcomings. They were time consuming, tedious and not precise enough (DeCarlo et al. 2011). The first LCM using primitive ultraviolet (UV) laser technology was established by Meier-Ruge et al. (1976). Another approach was published in 1993. Shibata (1993) described the destruction of DNA using an UV laser beam while cells were protected due to a specific dye. Unfortunately, this approach was only useful for metabolites that are susceptible to UV laser (DeCarlo et al. 2011). The 1990s seems to be a golden age of new LCM inventions. EmmertBuck et al. (1996) from the National Institutes of Health (NIH) developed an advanced LCM system with an infrared (IR) laser diode. Shortly thereafter, Schutze and Lahr (1998) introduced a new UV laser approach. Both techniques are described in Section 2.4.3. An overview of the development of laser Microdissection systems is given in Fig 16. Today’s LCM systems offer an extreme accurate, one-step-single process for preserving and isolating single cells or cell clusters from tissue sections (DeCarlo et al. 2011, Liu et al. 2014, Datta et al. 2015).

20

Methodological Principles

Fig 16: Timeline of Laser Microdissection Technology Development. Source: Espina et al. (2007).

2.4.2 Principles of Laser Capture Microdissection LCM is typically used to overcome drawbacks of heterogenous cell samples by isolating specific cell/tissue regions (Emmert-Buck et al. 1996, Espina et al. 2007, Ly et al. 2016). It enables the procurement of a microscopic and contamination-free cellular section away from its heterogenous tissue milieu (Lehmann et al. 2000, Liotta and Petricoin 2000, Liu 2010, Vandewoestyne et al. 2012, Hinneburg et al. 2017). Consequently, LCM facilitates the analysis of molecular alterations within a specific cell population (Cheng et al. 2013). Dissections can be taken from frozen or FFPE tissue blocks (Emmert-Buck et al. 1996, Liotta and Petricoin 2000) as well as from living cell culture dish plates. Microdissected tissues are generally used for downstream analysis (Espina et al. 2006). Many research groups have utilized LCM so far. Among them are DNA and ribonucleic acid (RNA) analysis, proteomics (Emmert-Buck et al. 1996) and molecular profiling, like point mutations or loss of heterozygosity (Lehmann et al. 2000). As mentioned in Section 2.4.1 the IR capture system and UV cutting system are two classes of LCM (Vandewoestyne and Deforce 2010) with their own advantages and disadvantages (Lehmann et al. 2000). LCM instruments are developed either as manual or automated system. The different techniques are described down below in Section 2.4.3.

2.4.3 Laser Capture Microdissection Techniques Fundamental components of an LCM system are: •

Inverted microscope (4 – 150-fold magnification), visualizing cells of interest.

•

IR and/or UV laser diode with a laser control unit.

•

Chuck for slide immobilization.

•

Microscope stage with charge coupled device camera.

•

Joystick for area selection and a color monitor (DeCarlo et al. 2011, Sauer 2015). 21

Methodological Principles

The first system developed by Schutze and Lahr (1998) is an UV laser microbeam microdissection system. Cells or areas of interest, respectively, are cut out of the tissue section by using a highly-focused laser beam (Fig 17). After the area of interest is cut properly, the laser power increases und catapult the desired cells against gravity into a collection device (Vandewoestyne et al. 2013). In this work, PALM CombiSystem (Zeiss) with an UV laser diode was utilized. The CombiSystem includes two non-contact laser technologies. The first one is PALM MicroBeam, which is applicable to cryo- and FFPE tissue sections and the second one PALM MicroTweezers which is mainly applied to living cell culture. The latter was not used in this work. The PALM MicroBeam was used to cut tissue/cell areas of interest from a polyethylene tetraphthalate (PET) membrane slide (Espina et al. 2006).

Fig 17: Schematic Representation of IR Laser (left) and UV Laser System (right). Abbreviations: IR = infrared; UV = ultraviolet. Source: Vandewoestyne et al. (2013).

Modern UV laser diodes focus the laser beam precisely in the same plane as the sample through the objective of the microscope (Sauer 2015). The laser pathway is shown in Fig 18. Cut areas are catapulted and captured accordingly by placing an adhesive cap very closely beyond the tissue section. Cells within the cutting path may sustain an UV-induced damage 22

Methodological Principles

(Espina et al. 2006). Nevertheless, the UV laser system has several advantages over the IR laser system. It allows faster and more precise sample collection. In addition, samples can be used more diversified (Vandewoestyne et al. 2013). This method is also schematically represented in Fig 17. The other system is also introduced. The stationary near-IR laser is mounted in the optical axis of the microscope stage. Selected cells of interest are adhered to a thin thermolabile polymer film in a fixed position (Vandewoestyne et al. 2013). The cap of a tube is placed in the same plane as the sample and used as an optic to focus the laser (Espina et al. 2006). The laser melts the thermolabile polymer and removes it with the underlying cells from the tissue surface (Espina et al. 2006, Vandewoestyne et al. 2013). The IR laser pulse duration is short, and thus, only low laser powers are needed. The laser elevates the temperature on the membrane temporarily until 90°C, but only cells right next to the laser are affected (Wernert 2004). The reason is an incorporated dye into the polymer which absorbs the laser energy. Hence significant heat cannot be generated and the LCM procedure does not result in significant denaturation of biomolecules. But the diameter of an IR laser beam is between 7.5 − 35 µm (Liotta and Petricoin 2000, Cheng et al. 2013) and therefore, far thicker compared to an UV laser (≤1 µm) (Cheng et al. 2013). A schematic representation of the IR laser technique is shown in Fig 17.

Fig 18: LCM system PALM Microbeam (Zeiss). Blue lines represent the beam path of the UV laser and yellow lines the bright field illumination upon the object. Abbreviations: LCM = laser capture microdissection; PALM = photoactivated localization microscopy; UV = ultraviolet. Source: Sauer (2015), page 73.

23

Methodological Principles

2.5

N-Glycan Processing

2.5.1 Enzymatic N-Glycan Release Glycans can be released by chemical or enzymatic methods (Wuhrer et al. 2009b). Chemical approaches are base-catalyzed β-elimination or hydrazinolysis. The enzymatic release is attained by using peptide:N-glycanase (PNGase) F, PNGase A, glycosaminidase or endoglycosidase like endo-F and endo-H (Merry and Astrautsova 2009). For most N-glycans PNGase F [EC 3.5.1.52] will be the enzyme of choice (Hirayama et al. 2015, Kolarich et al. 2015). PNGase F hydrolyses the β-aspartylglycosylamine bond between GlcNAc and Asn (Rudd et al. 2001). However, glycans containing an α1,3-Fuc core shall be treated with PNGase A (Tretter et al. 1991, Kuster et al. 1998). Furthermore, N-glycans attached to Asn residues in terminal position are barely cleaved by PNGase F. But PNGase A is still not the deglycosylation enzyme of choice for mammalian glycoproteins since it has some disadvantages. For example, PNGase A does not act on intact glycoproteins. With its molecular mass of 75.5 kDa is PNGase A much larger than PNGase F (36 kDa). Thus, penetration of PNGase A into gel could be hindered (Kolarich and Altmann 2000). The cytoplasmic enzyme PNGase F is involved in the degradation of misfolded glycoproteins (see ERAD in Section 1.4.2). Its optimal pH is essentially neutral. N-glycan release through PNGase F can be divided into two steps. First, N-glycans on the consensus sequence Asn-XSer/Thr (X is any AA except Pro) are hydrolyzed by PNGase F (Hirayama et al. 2015, Zhang et al. 2015). This reaction leads to the formation of Asn to aspartic acid (Asp) and a free glycan with a 1-amino-GlcNAc. In the second step, ammonia from 1-amino-GlcNAc is nonenzymatically released remaining GlcNAc at the reducing termini of the glycan (Hirayama et al. 2015). The reaction mechanism of PNGase F is shown in Fig 19. A wide range of different buffers were applied for PNGase F application. The most common buffer is ammonium bicarbonate (NH4HCO3) in concentrations between 25 mM and 1 M and a pH range from 7.5 – 8.5 (Stumpo and Reinhold 2010, Ritamo et al. 2013, Toghi Eshghi et al. 2014, Turiak et al. 2014, Gustafsson et al. 2015, Kolarich et al. 2015, Everest-Dass et al. 2016, Hecht et al. 2016, Zhou et al. 2017). Other buffers can be used as well. Sodium phosphate was applied in a concentration of 50 mM with pH 7 (Jeong et al. 2012) and pH 7.5 (Hu and Mechref 2012). Donczo et al. (2016) performed deglycosylation in sodium bicarbonate (pH 7). Phosphate buffered saline 2 – 5x concentrated in combination with 2 – 4% NonidetTM P-40 is also applicable (Ruhaak et al. 2008, Selman et al. 2011). Buffer preparations with Tris are used as well (Burnina et al. 2013). As it becomes challenging to analyze N-glycans in presence of hydrophobic proteins and peptides, N-glycan release is followed by purification from salts, detergents, proteins, and peptides. Hydrophobic proteins and peptides are more efficiently ionized by MS and on top of that they suppress the ionization of glycans. Unmodified glycans have only little or no retention on hydrophobic absorbents (Yang and Zhang 2012). Therefore, reverse phase (RP) solid phase extraction (SPE) is used to isolate the glycans in the protein/peptide mixture. Common

24

Methodological Principles

hydrophobic absorbents for N-glycan purification are C8 (Aldredge et al. 2012) and C18 (Kuster et al. 1997, Stumpo and Reinhold 2010) columns, cartridges or tips.

Fig 19: N-Glycan Release Catalyzed by PNGase F. Abbreviations: Asn = asparagine; Asp = aspartic acid; GlcNAc = N-acetylglucosamine Source: Hirayama et al. (2015), page 112.

2.5.2 Fluorescence Labeling of N-Glycans Glycans have usually low intrinsic spectral activity and because of that detection of glycans through chromatographic processes may require a labeling technique. An extremely useful tool is fluorescence labeling as it allows to monitor very low concentrations of glycans (France et al. 2000). Fluorescent labeling by 2-aminobenzamide (2AB) and 2-aminobenzoic acid (2AA) on a nonselective manner through reductive amination was first introduced by Bigge et al. (1995). Reductive amination requires a free reducing terminus on the glycan (Merry et al. 2002, Wuhrer et al. 2009b). The reaction is shown in Fig 20. Derivatization with 2AB take place at C1 at the very first GlcNAc which was attached to its protein (Rudd et al. 2001). The conjugation per se is mostly independent of the glycan concentration. Reducing of glycans is mediated through sodium cyanoborohydride (NaBH3CN) (France et al. 2000). The stoichiometric attachment of one labeling molecule per glycan allows the relative quantification of glycans

25

Methodological Principles

based on fluorescence intensity (Kozak et al. 2015). This is ensured through excessive addition of fluorescence tags, resulting in a large amount of free label reagent (Merry and Astrautsova 2009) to be removed afterwards (see Section 2.5.3). Interestingly, the required reaction temperature (~ 65°C) does not lead to significant losses of SiaAc (both, α2,3- and α2,6-linkages) which is the less stable sugar component of a glycan. 2AB labeling has also been established for milk oligosaccharides (Marino et al. 2011) and bovine whey protein (van Leeuwen et al. 2012). The 2AB label is compatible with most chromatographic and MS methods while 2AA is well-suited for electrophoretic techniques, but not for RP-HPLC, MS or ion-exchange chromatography (Bigge et al. 1995). On the other hand, the fluorescent intensity of 2AA is more than twice as high as 2AB (Anumula and Dhume 1998). Fluorescence labeling is not applicable to glycans released under reductive conditions as the required aldehyde group is reduced to an alditol (Zaia 2008).

Fig 20: Reaction of Reductive Animation by Fluorescence Tags. Source: Han and Costello (2013), page 713.

Other reagents like 8-aminonaphthalene-1,3,6-trisulphonic acid, 2-aminopyridine (2AP), 4aminobenzoic acid (Yuen et al. 2002), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), procaine, procainamide (Klapoetke et al. 2010, Kozak et al. 2015), 2-aminoacridone (2AmAc) (Knezevic et al. 2011) and aminoquinoline (AQ) (Struwe and Rudd 2012) are some other popular examples, with their own advantages and disadvantages.

2.5.3 Purification of Excessive Labeling Reagents After derivatization with 2AB, purification of excess labelling reagent prior to chromatography should be considered (Benet and Austin 2011) in order to avoid detergent clusters. No purification would cause difficulties in separating peaks from small glycans to the relatively non-hydrophobic label reagent (Merry and Astrautsova 2009). In contrast to the first purification step (Section 2.5.1), derivatized glycans show high retention on NP absorbents (Yang and Zhang 2012). Appropriate techniques for this purpose are, paper chromatography (Merry and Astrautsova 2009), hydrazine coupling, lectin antibody chromatography, and HILIC SPE. Especially HILIC SPE is particularly useful for the enrichment of N-glycans and may be preferred for glycomic analysis (Selman et al. 2011). Acetonitrile (ACN) concentrations around 80% lead to retention of glycans while elution is achieved with higher water content (Wuhrer et al. 2009a). 26

Methodological Principles

2.6

High Performance Liquid Chromatography

Glycans can be separated on the basis of specific properties, like charge or hydrophilicity which is under certain conditions equivalent to mass (Merry and Astrautsova 2009). NP-HPLC on amide-based columns as well as RP-HPLC have been considered as useful method in the characterization and sequencing of glycans of biological interest (Merry and Astrautsova 2009, Gil et al. 2010, Vreeker and Wuhrer 2017). NP-HPLC utilizes polar stationary phase and nonpolar mobile phase and represents roughly the opposite retention pattern of RP-HPLC (Aguilar 2004, Mysling et al. 2010). Even if RP-HPLC systems were commonly used by scientists, NPHPLC methods started to undergo a renaissance a couple of years ago (Buszewski and Noga 2012). In this work, zwitterionic (ZIC)-HILIC-HPLC, a variation of NP-HPLC, was used for N-glycans separation. The principle of ZIC-HILIC is based on a silica-based zwitterionic stationary phase with both, negative (sulfonic acid) and positive charges (quaternary amine). Glycans are retained on the hydrophilic stationary phase through ionic interactions (Mysling et al. 2010), hydrogen bonding and dipole-dipole interactions (Wuhrer et al. 2009a). This clarification points out that the separation mechanism of HILIC is far more complicated than that in NP-HPLC (Buszewski and Noga 2012). In order to avoid confusion, HILIC was previously described as a feasible SPE technique for the enrich glycoproteins or glycans (Section 2.5.3). ZIC-HILICHPLC is from now on referred as HILIC while its usage for purification is named as HILIC SPE. HILIC combines advantages of conventional NP- and RP-HPLC. It is suitable to analyze samples that elute near the cavity of RP-HPLC. Polar samples show beneficial solubility in aqueous mobile phase used in HILIC, and therefore, it overcomes the drawbacks of poor solubility in NP-HPLC systems. Uncharged highly hydrophilic and amphiphilic samples which are too polar to be retained appropriately by RP-HPLC or ion-exchange chromatography can be separated well by HILIC. Furthermore, HILIC systems can be conveniently coupled to MS, especially ESI-MS. Taken together, HILIC employs polar stationary phases like NP-HPLC, but the mobile phase is similar to RP-HPLC. It also allows the analysis of charged substances like ion chromatography (Buszewski and Noga 2012). The characteristics of HILIC are shown in Fig 21. Typical mobile phases for HILIC are water-miscible polar organic solvents like ACN (Buszewski and Noga 2012) and an ammonia salt dissolved in water like ammonium formate (NH4HCO2) (Guile et al. 1996), NH4HCO3 (Kolarich et al. 2015) or ammonium acetate (NH4CH3CO2) (Buszewski and Noga 2012). The following eluotropic row lists some solvents according to increasing elution strength in HILIC: acetone < isopropanol < ACN < ethanol < dioxane < methanol< water. HILIC separation can be performed in isocratic mode with high percentage of organic solvent or with gradient mode starting with a high percentage and ending with low portion of the organic solvent (Ahn et al. 2010, Buszewski and Noga 2012).

27

Methodological Principles

Fig 21: Combination of Different Chromatography Characteristics in HILIC. Abbreviations: HILIC = hydrophilic interaction chromatography; IC = ion chromatography; NP-LC = normal phase liquid chromatography; RP-LC = reversed phase liquid chromatography. Source: Buszewski and Noga (2012), page 232.

Due to its advantages, HILIC has been widely used to analyze derivatized glycans. However, HILIC favors N-glycans over O-glycans since N-glycans are usually larger and therefore, more hydrophilic than O-glycans (Mysling et al. 2010). One disadvantage of HILIC is its incapacity to observe the resolution of structural isomers (Zaia 2008). ACN is the most commonly used organic solvent for N-glycan analysis by HILIC. Even if N-glycans elute mostly with descending percentage of ACN, time or concentration, cannot be predicted without testing. Farther, elution of N-glycans depends on pH, temperature, column type, column size and gradient adjustment (Kolarich et al. 2015). Especially the solvent pH has a significant effect on HPLC separation of sialylated N-glycans. For example, the dissociation of SiaAc is suppressed at pH 2.5 as the pK of SiaAc is 2.6 (Guttman et al. 1996). Finally, chromatographical approaches require a single detection system. This can be either an electrochemical detector (HPAEC-PAD) or a fluorescence detector (Merry and Astrautsova 2009). The latter was used in this work as N-glycans were fluorescent labeled through 2AB.

2.7

Permethylation

Permethylation defines complete methylation of all hydroxyl groups within a glycan (Wallenfels and Bechtler 1963, Price 2008) as shown in Fig 22. Permethylation allows rapid and reproducible screening of sialylated glycans (Reiding et al. 2014) and is an important step prior MS analysis (Asres and Perreault 1997, Kang et al. 2008). Its most significant feature is the conversion of SiaAc to methyl ester and hence the neutralization of multiple negative charges carried by sialylated N-glycans (Yu et al. 2006, Wheeler et al. 2009). SiaAc would be otherwise both, unstable during its ionization and has a low positive mode ionization efficiency. The carboxyl group present on SiaAc preferentially facilitates negative ionization by MALDI-MS, and this could bias the quantitative measurement of sialylated glycans when comparing acidic and neutral glycans (Reiding et al. 2014, Zhou et al. 2017). Sample pre-treatment by permethylation supplies mass spectra of better quality with an enhancement of signal to noise 28

Methodological Principles

ratio for both, MALDI-MS and ESI-MS, respectively (Yu et al. 2006). Permethylation yields more predictable ion products in sialylated glycans subjected to tandem MS (MSn) experiments (Kang et al. 2005), shown in Section 2.8.3. Directional fragmentation of permethylated glycans allows better interpretation of structure and branching information (Price 2008) and easy determination of interglycosidic linkages as well as conformational and configurational isomers (Kang et al. 2005).

Fig 22: Reaction of Permethylation. Source: Han and Costello (2013), page 713.

The most common used permethylation protocol is introduced by Ciucanu and Kerek (1984), which is based on the use of dimethyl sulfoxide (DMSO), methyl iodide (CH3I), and solid sodium hydroxide (NaOH). This reaction is sensitive to the presence of water. That is why the reaction is undertaken in dry solvent like DMSO (Price 2008). DMSO removes protons from the sample molecules prior to substitution with methyl groups. The original procedure of Ciucanu and Kerek (1984) was modified a couple of times resulting in experimental simplicity, purer reaction products and rapidity (Kang et al. 2005). Permethylation provides a simple way of glycan purification from its buffers by imparting them hydrophobic nature and therefore, conductive to RP-SPE type of purification (Yu et al. 2006). But permethylation is not compatible with some glycan modifications like sulfate or phosphate groups, respectively (Zaia 2008). For PNGase F released glycans, mass differences through permethylation have to be calculated accordingly. Every hydroxyl group can be methylated resulting in methyl-glycoside (CH3O) with a sodium giving the positive charge. Methylation leads to the addition of 14 Da to every former hydroxyl group (Morelle and Michalski 2007). Lists of mass differences are given in the Appendix (Tab 26 and Tab 28).

2.8

Mass Spectrometry

MS provides because of its resolution, speed, and sensitivity proper opportunities for glycomic analysis including structural characterization of protein glycosylation, determination of sitespecific glycosylation as well as analysis of N- and O-glycans (North et al. 2009, Selman et al. 2011, Kolarich et al. 2015, Zhou et al. 2017). MS of 2AB labeled N-glycans has been performed on deprotonated species (negative-ion mode), as well as on proton adducts and sodium adducts (positive ion mode). With permethylation for sialylated glycans (Section 2.7) positiveion mode yields the most stable results. Most widely distributed MS instruments for the analysis of 2AB labeled (and permethylated) glycans are quadrupole time of flight (TOF) and ion traps (Wuhrer et al. 2009b). 29

Methodological Principles

MS has the ability to specify an unequivocal molecular mass. However, sequence information may need tandem MS (MSn) (Fu et al. 1994, Zhou et al. 2017). But the characterization of proton/sodium adducts by MSn with collision-induced dissociation (CID) or laser-induced dissociation (LID) leads mainly to B- and Y-type fragments (Wuhrer et al. 2006, Wuhrer et al. 2009b). Carbohydrate fragmentations are shortly discussed in Section 2.8.3. Common encountered B-type ions are to be found in the Appendix in Tab 27. Another very appropriate possibility for N-glycan sequencing is the use of linkage specific exoglycosidase (Morelle and Michalski 2007) which is briefly described in Section 2.8.4. Nonetheless, efficient ionization and detection of glycans may need proper purification steps (Selman et al. 2011) as described in Sections 2.5, 2.5.2, and 2.7.

2.8.1 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry MALDI-TOF-MS is a well-suited technique for high-throughput glycomics (Kolarich and Altmann 2000, Hung et al. 2012, Jeong et al. 2012, Reiding et al. 2014). It has a relatively high tolerance to salts and other contaminants (Zaia 2008). MALDI-based MS profiling is often the preferred first step in N-glycan analysis as it take less time compared to HPLC-ESI-MSn (Yu et al. 2006, Morelle and Michalski 2007). Glycans are composed of a relatively small number of various monosaccharides constituents (Section 1.2.2) with unique incremental masses (Appendix Tab 26). That is why profiling of permethylated glycans using MALDI-TOF-MS allows to obtain information about nature and diversity of glycan structures rapidly by applying multiple samples on a metal target simultaneously. The signal strength of permethylated glycans reflects accurately the relative quantities of glycans (Morelle and Michalski 2007). MALDI-TOF-MS seems to lack in detection of lower abundant N-glycan structures. To improve the identification and quantitation of low abundant glycans MS can be coupled to other separation techniques. Hydrophobic permethylated glycans are efficiently separated with RPHPLC (Karlsson et al. 2008, Stavenhagen et al. 2017, Zhou et al. 2017). MALDI-TOF-MS is used best for analysis of chromatographic fractions which are collected offline (Zaia 2008). The most prosperous approach is an online separation step before MS analysis by using the combination of RP-HPLC-ESI-MS (Hu and Mechref 2012) as discussed in Section 2.8.2. Samples need to crystalize on a metal target prior analysis. It has been stated that 2,5dihdroxybenzoic acid (2,5-DHB) is a suitable matrix for MALDI-TOF-MS measurement of carbohydrates (Harvey 2006, Harvey et al. 2012, Hung et al. 2012). It produces [M + Na+] species as the major ion (Harvey 1999). However, for the MALDI-TOF-MS studies, glycerol prevents the co-crystallization of the 2,5-DHB matrix (Morelle and Michalski 2007) for which reason PNGase F (Section 2.5.1) should be obtained in glycerol-free form. Formalin does not affect MALDI-MS analysis (Dwek et al. 1996)

2.8.2 Electrospray-Ionization Mass Spectrometry ESI-MS entails spraying a solution containing the sample through a needle to which an electrical potential is applied. This creates a condition in that very fine droplets are formed. Depending on their charge droplets are either attracted toward the needle or repelled. Appropriate charged droplets move toward the MS orifice while undergoing solvent 30

Methodological Principles

evaporation. This leads to the formation of multiple charged gas phase ions which are analyzed in the MS (Zaia 2008). ESI-MSn with low energy CID is a valuable tool for the production of fragment ions to analyze glycan sequence and even linkages (Morelle and Michalski 2007) especially for permethylated sialylated N-glycans (Kang et al. 2005). The extent of fragmentation of sialylated glycans is quite lower than observed using MALDI-MS (Zaia 2008). Different to MALDI-MS, samples should be free of salts prior to injection into the ion source. Otherwise multiple cation adduction may appear. Hence, HPLC is performed previously to ESIMS analysis, either offline or online (Zaia 2008).

2.8.3 Fragmentation of Glycans Fragmentation of glycans depends on several factors, like type of ion formation, its charge state and energy deposited into the ion. As a rule, two major types of ions are usually observed (Morelle and Michalski 2007). First, glycosidic cleavages where the bond is ruptured between the sugar rings. The reducing end of the original glycan can be maintained through the production of an ion (Y and Z fragments) or not (B and C fragments). The CID chamber can also induce the cleavage of the carbohydrate ring provoked by breaking two bonds. Those fragments resulting from cross-ring cleavages can maintain the reducing end (X fragment) or not (A fragment). Taken together, A, B and C fragments can retain the charge on the nonreducing end while X, Y and Z fragments retain the charge on the reducing end. A and X result to cross-ring cleavages, while B, C, Y and Z correspond to glycosidic cleavages (Zaia 2004, Morelle and Michalski 2007, Ceroni et al. 2008). The presented nomenclature has been devised by Domon and Costello (1988). Fragmentations are shown in the Appendix in Fig 49. Permethylated glycans yield intense sodium-cationized and protonated species (Morelle and Michalski 2007). Sodium adducts usually reveals patterns of ring fragmentations which are characteristic for linkage positions (Wuhrer et al. 2006). Fragmentation of permethylated glycans result in B and Y fragments. B fragments (Tab 27) result predominately from cleavage of the glycosidic bond at the GlcNAc moiety with location of the charge on the non-reducing terminus. Such fragment ions are extremely useful for antennae sequence allocation (Morelle and Michalski 2007).

2.8.4 Exoglycosidase Digestion Definition of monosaccharide sequencing and their anomeric configurations can be achieved by treatment with exoglycosidases. These enzymes remove monosaccharides from the nonreducing end of the glycan chain. Exoglycosidases are specific to stereochemistry, the anomeric configuration to the released monosaccharide and its linkage bond with regard to the remaining monosaccharide on the glycan chain. It is recommended to use highly specific exoglycosidases in a reasonable order, starting with α-sialidase, followed by β-galactosidase, β-N-acetylglucosminidase, α-fucosidase and α-mannosidase digestion. After each step, the mixture is analyzed by MS. Mass changes after each digestion step reveals the sequence of the glycan. MALDI-TOF-MS is may be preferred (Morelle and Michalski 2007) as it allows rapid screening (Section 2.8.1). Tab 29 (Appendix) shows a list of commonly used exoglycosidases for glycan sequencing. 31

Methods

3

Methods

Note: Room temperature (RT) was considered as 18 – 22°C and overnight incubation lasted approximately 16 h. Pipettes and pipette tips were not listed in any material list. All tables listing required materials are to be found in the Appendix in Section 8.1.

3.1

Preparation of Standard Glycans

3.1.1 Preparation of Dextran Hydrolysate Dextran hydrolysates were prepared as described in Wuhrer et al. (2009b). Materials required for hydrolysation of dextran are listed in Tab 5. Dextran hydrolysate was used as a standard glycan in order to determine an optimal HPLC gradient. 10 mg mL−1 dextran was dissolved in 0.1 M trifluoroacetic acid (TFA) in a HPLC glass vial. Samples were sonicated for 3 min and then incubated for 1 h at 100°C in a thermo shaker. The Calculation for the preparation 0.1 M TFA is explained in the Appendix in Section 9.4.1. Approximately one tenth (~ 100 µL) was transferred into a 1.5 mL tube and dried in a centrifugal evaporator. The dried sample was dissolved in 100 µL 2AB solution and labeled as described in Section 3.5.4. After fluorescent labelling, additional 1 mL of Milli-Q®-water was added and samples were sonicated again for 3 min. At last, samples were stored either at 4°C or at −20°C.

3.1.2 Preparation of Standard Glycoproteins Materials required for the preparation of standard glycoproteins are listed in Tab 6. Used standard glycoproteins were mentioned in Sections 2.2.2, 2.2.3, and 2.2.4. Standard glycoproteins were dissolved in 25 mM NH4HCO3 (pH 8.0). NH4HCO3 was used throughout many protocols (Sections 3.4.1, 3.4.2, 3.5.1, and 3.5.2) and its preparation and calculation is explained in the Appendix (Section 8.5.2). NH4HCO3 was not stored longer than two weeks as it changes its pH value over time (Hinneburg et al. 2017). Hereafter, 200 µg of standard glycoprotein was transferred in a 0.5 mL tube and dissolved in 50 µL of NH4HCO3. For proper solution of glycoproteins, samples were sonicated for 3 min in an ultrasonic bath. The protocol was either continued in Section 3.4 or samples were stored at −20°C until usage.

3.2

Preparation of Formalin-Fixed Paraffin Embedded Tissues

Protocols for the preparation of FFPE tissues are highly standardized in different labs. The protocol used in this thesis was adapted from PD Dr. Sebastian Galuska’s Group (Leibniz Institute Dummerstorf), Prof. Dr. Ralf Middendorf’s Group (Justus-Liebig University Giessen) and some current publications (Stoehr et al. 2003, Casadonte and Caprioli 2011, Ly et al. 2016, Hinneburg et al. 2017) First, FFPE tissue blocks have to be sectioned and mounted on histology glass slides (Section 3.2.1) and thereafter deparaffinized and stained (Section 3.2.2). An additional approach of deparaffinization in tubes without mounting on slides is also described (Section 3.2.3).

32

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3.2.1 Cutting Procedure Materials required in order to cut slides from FFPE tissue blocks are listed in Tab 7. Before starting, tissue blocks were stored at −20°C for 30 min to optimize the cutting procedure. While tissue blocks were incubating in the freezer, the microtome was prepared (e. g., filling the water bath with distilled water, and setting the blade into its slot). Slits were cut into in the paraffin range next to the tissue (Fig 23B) with the intent to attain smaller sections. Therefore, more sections could be mounted on each slide and less paraffin had to be removed afterwards. Prepared FFPE tissue blocks were sectioned in 5 µm thick slides using a microtome (Fig 23A). Slides were then further processed in two different ways. First, samples were directly transferred into (LoBind) tubes and also deparaffinized in them. In this case, the protocol continues in Section 3.2.3. The second approach includes mounting tissue sections on either histology glass slides (analysis of whole tissue sections) or PEN slides (LCM).

Fig 23: Used Microtome for Tissue Sectioning. A = Microtome obtained by Leica; B = paraffin blocks were slit next to the tissue which led to smaller sections to be mounted on the histology glass slide; C = the blade has to be put very carefully into its slot; D = cold water container used to gather tissue slides; E = warm water bath was used to stretch and align sections on the glass slide; F = histology glass slides were put onto a heating plate.

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Slides were gathered in a container filled with cold distilled water (Fig 23D) and then mounted onto glass slides. Tissue sections were mounted on histology glass slides for analysis of whole tissue section or visualization of morphological/histological characteristics. In case of LCM analysis, the first cut was mounted on a PEN slide and every following section was mounted on histology glass slides in order to count sectioned cells or scouting appropriate areas for microdissection, respectively. Next, glass slides were dunked into the heating bath (Fig 23E), filled with distilled water at 37°C. After the sections had been stretched appropriately, tissue sections were aligned back on its glass or PEN slide. Finally, histology or PEN slides were put on the heating plate at 37°C until all sections were prepared. The used apparatus is shown in Fig 23. Complete dying of all sections was accomplished in a heating oven for at least 4 h at 37°C (Fig 23F). Dyed sections can be stored at RT until usage (Casadonte and Caprioli 2011). The protocol for deparaffinization and staining is continued in Section 3.2.2.

3.2.2 Deparaffinization and Staining on PEN and Histology Glass Slides Deparaffinization and staining (optionally) was usually performed on the next day. Materials required for deparaffinization and HE staining are listed in Tab 8. Histology glass or PEN slides were set in a coplin rack and heated in an oven near to the melting point of paraffin (~ 50 – 60°C) for approximately 60 min. Prior heating optimizes deparaffinization by saving treatment time in organic solvents. During the heating process coplin jars were filled with Roti®Histol (twice), 100% (v/v) isopropanol (thrice), 70% (v/v) isopropanol (twice), 50% (v/v) isopropanol (twice), distilled water (once), Hemalaun (once), and eosin (once). When the heating procedure was finally finished, coplin racks were immersed in Roti®-Histol for 5 min. This procedure was repeated once in fresh Roti®-Histol for additional 5 min. Samples for LCM were washed and carefully rehydrated in 100%, 70% and 50% isopropanol for 3 min each and then immersed in distilled water for 3 min before staining. If complete tissue sections were supposed to be investigated, the rehydration procedure was skipped. Staining would be redundant as air-dried tissue sections were scraped off using a razor blade and transferred into 1.5 mL tubes anyway. Tissue sections were scraped off without wearing nitrile gloves in order to avoid static repulsion. The use of a surgical mask with the purpose of not breathing tissue sections away was beneficial (Hinneburg et al. 2017). The protocol for the analysis of whole tissue sections was continued as described in Section 3.4. If the experiment was paused, samples were stored at −20°C until usage. Here, the protocol continues for samples to be stained, including LCM approach and their corresponding follow cuts. Hemalum was applied for 3 min followed by washing under running distilled water for 3 min. Optionally, eosin staining can be used afterwards. The procedure applied was similar and differed only in time (1 min each). After staining, tissue sections on PEN membrane slides for LCM approach were dehydrated again in a reversed order. Membrane slides were immersed in 50%, 70%, and 100% isopropanol for 3 min each. The dehydration causes flatten of cells. Deparaffinized and dehydrated tissue sections were airdried before usage. Protocol was continued as described in Section 3.3. Corresponding follow cuts for microscopical approach must not be dehydrated. In order to maintain structural 34

Methods

morphology mounting medium was used (Riedelsheimer and Buchl-Zimmermann 2015). One drop of Roti®-Mount Aqua was given on the tissue section and then covered with cover slips while air bubbles were displacing carefully with a pin. Mounted and covered tissue sections were air-dried and stored at RT.

3.2.3 Deparaffinization in Tubes Deparaffinization of whole tissue sections was also accomplished in tubes. Decisive advantages are less solvent consumption and samples loss through scratching, but the protocol is far more time consuming. Materials needed are listed in Tab 9. This protocol was mainly adapted from Patel et al. (2016). Tubes, containing tissue sections were shortly spun using a micro centrifuge at a fixed speed of 7,000 rpm. Next, tubes were incubated at 50°C for 3 min. One milliliter Roti®-Histol was added and tubes were vigorously vortexed for 10 s. Tubes were centrifuged at RT at highest speed possible (here 13,000 rpm) followed by incubation on ice for 5 min. Cooling down allowed waxy residues to solidify on the top, shown as paraffin accumulation around the meniscus. Supernatants were carefully removed accordingly. This procedure was repeated once again with Roti®-Histol and twice with 100% isopropanol. Samples were shortly allowed to air-dry by opening the caps of each tube. The protocol was continued in Section 3.4 or samples were stored at −20°C until usage.

3.3

Laser Capture Microdissection

In this protocol, areas of interest were laser captured followed by further preparation for bioanalytical approaches as described in Sections 3.4, 3.5, 3.6, 3.7, and 3.8. Specific cell types were selected using photoactivated localization microscopy (PALM) RoboSoftware according to manufacturers’ (Zeiss) manual. The whole LCM device used is shown in Fig 24. Materials used in this Section are listed in Tab 10. Preparing and using the LCM device is not as trivial as one might assume. All devices were switched on and the PALM Robo Software was accessed. The laser unit will always take the most time until the operating temperature is reached. It is important to note, that all steps described in this protocol should be performed one after the other. Components moving towards each other will neither recognize counterparties nor stop automatically. In the worst case, they run against each other leading to damage and incurring significant costs. The TubeCollector was loaded with AdhesiveCaps and then inserted into the CapMover (Fig 26A and Fig 26B). Next, up to three prepared tissue sections on the PEN slides were placed on the SlideHolder. This step must not be accomplished on the load position as pressure could damage the device (Fig 26A). To ensure proper cutting, the LCM components have to be adjusted first. Adjustment steps were mostly performed using the PALM Robo Software. The load position was driven out by clicking “Move to Load Position”. The SlideHolder with PEN slides was carefully placed into its mount and the load position was driven back by clicking “Return to Working Area”. Objective was set to 20-fold magnification. The camera included illumination and focus were set either via “Auto Live” for live adjusting or by “Autoconfig” for adjusting on command. The live version is easier to handle, but in return it slows down the processing capacity. Brightness was set to maximum (3200 K). 35

Methods

Fig 24: Used LCM Device in this Work. PALM CombiSystem including PALM MicroBeam (cryosections and FFPE tissue sections) and PALM MicroTweezers (living cell culture) combines two non-contact laser technologies within one system. Note: Only PALM MicroBeam was used in this protocol. Abbreviations: FFPE = formalin-fixed paraffin embedded; LCM = laser capture microdissection; PALM = photoactivated localization microscopy.

The image contrast was set by opening or closing the aperture diaphragms set manually (no part of PALM Robo Software). Thereafter, both AdhesiveCaps were adjusted in two steps with “Cap Mover II”. First step, caps were set wafer-thin (~ 0.5 mm) over the tissue section as shown in Fig 26A. Second, by using the function “Cap Check”, AdhesiveCaps were exactly positioned over the objective. This step ensured capturing of microdissected cells preferably in central position of the AdhesiveCap. The laser was adjusted last. If required, the objective was focused once more. The laser is adjusted appropriately when laser and cursor are matched. First, “Cut Laser” was chosen as “Laser Mode”, and “Dot Mode” as “Cut Type”. A single dot was drawn on a PEN membrane-only are by using the Smart Stylus on the touch screen. Then, depending on configuration/version, “F2”, “F7” or “F11” button on the keyboard was typed for starting synchronization or laser adjustment, respectively. At this point it is important to avoid any movement of the cursor. The laser was activated by pressing a foot pedal and synchronized/laser adjustment was finalized by typing “F2”, “F7” or “F11” again. Proving whether the adjustment had worked or not, “Line Mode” was chosen to draw a sinuous line with the Smart Stylus on the touch screen. The line should be drawn again on the PEN membrane-only. Down left, the laser mode was set on “Cut” and “Manual”. The laser was started by clicking the sandglass with the crosshair inside it (“Start Laser Function”). If the laser was cutting the drawn line, laser power and laser focus were set. Otherwise, the adjustment procedure was repeated. A second sinuous line was drawn for the adjustment of laser power and focus. This time, however, the cutting procedure was performed on the tissue area. Laser power and focus needed to remove areas of interest differs in each tissue or cell type. It is important to note, that the adjustment has to be carried out while the laser is cutting. 36

Methods

Therefore, while the laser was cutting through the sinuous line, the laser power and focus were set by changing the scale for “Energy” and “Focus” accordingly. For the laser power that means surrounding cells at the cutting line should not be corrupted but still, the laser needs to cut throughout the tissue to ensure that cells/area of interest can be removed and captured. Adjusting the focus sharpens the laser cut but prevents the PEN slide and tissue from stoving by the laser. In this work, appropriate settings for epididymis (roe deer, mice, and rat) as well as lung (mice and rat) were 45 – 55 for “Cut Energy”, and 76 – 82 for “Focus”. Besides, cut areas need to reach the AdhesiveCap. For that, the laser pressure catapulting “LPC” was adjusted. Its “Energy” called “Delta” is usually set between 20 – 30, however, differences between tissue sections should be considered. The laser focus has also an adjustable “Delta” which was not changed during this work (setting was −2).

Fig 25: PALM Robo Software 4.5 Pro Surface. Abbreviations: PALM = photoactivated localization microscopy.

37

Methods

Before the actual cutting procedure started, the laser mode was set on “RoboLPC” and “Tube” (either Tube 1 or Tube 2). “RoboLPC” automatically combines cutting and catapulting even if the marked area is not enclosed. Last important settings included “Cutting Iteration” which ensures how often a section was cut successively and “Speed Percentage” which was usually in the lower range (15 – 35). All bold marked steps are shown in Fig 25. After finishing the whole adjustment procedure, the actual cutting procedure started by selecting cells/areas of interest by using the Smart Stylus. Using “Rectangle Mode” or “Circle Mode” was renounced. Areas were chosen by using “Line Mode” by drawing compatible lines along tissues of interest.

Fig 26: Schematic Representation of LCM Protocol. A = Load position in work area and adjusted AdhesiveCap; B = mounting AdhesiveCaps into the TubeCollector; C = selected cells of interest, here sperms from roe deer epididymis; D = microdissected cells form C; E = microdissected cells adhered to AdhesiveCap; F = not complete detached areas and use of single dots (blue) to release them. Abbreviations: LCM = laser capture microdissection.

38

Methods

After selecting cells of interest, pictures were taken by clicking the photo camera sign (“Save Image”) as shown in Fig 26C. Next, cutting and catapulting procedure were started by clicking the sandglass with the crosshair inside it. The magnification was usually set to 20-fold. Cutting procedure was repeated until all sections were detached. If sections did not detach at all (Fig 26F), single dots (“Dot Mode”) were plotted on not intersected areas leading to additional catapulting power right on the spot. When all sections were removed (Fig 26D), another picture was taken of the cut areas. Furthermore, the AdhesiveCap containing all tissue sections was photographed, too. To be sure that all cut areas were catapulted accordingly, magnification was set to 5-fold and the tissue area was searched for remaining sections. If sections were found, the dot procedure as described was repeated on the dissected tissue section. As soon as sufficient sections were gathered, the AdhesiveCap was taken out of its position by clicking “Change Collector”. AdhesiveCap was closed immediately to avoid losing any microdissected fragments (Fig 26E). Finally, the “Element List” was opened and all data of each sample was imported as text file. Before starting with the next sample, the element list was deleted and renumbered. Lids of AdhesiveCaps were supplementary enclosed with parafilm. Microdissected samples were stored at −20°C until usage. The protocol was continued in Section 3.4.

3.4

Antigen retrieval

3.4.1 Trypsin Mediated Antigen Retrieval The first approach used the serine protease trypsin [EC 3.4.21.4] which hydrolysis peptide bonds predominantly after positively charged Lys and Arg at the carboxyl site (Polgar 2005). Used materials for tryptic mediated antigen retrieval are shown in Tab 11. Trypsin solution (1 mg mL−1) in 0.001 N HCl was prepared first (see Appendix Section 8.5.3). One microliter of trypsin solution was dissolved in 100 µL NH4HCO3 (Sections 3.1.2 and 8.5.2) and then transferred to each sample. In case of microdissected tissue sections, more preparation steps were required which are described in Section 3.4.2. As standard glycoproteins were already dissolved in 50 µL of NH4HCO3, 1 µL trypsin solution was dissolved in the half amount of NH4HCO3 and then transferred into the sample tube. Samples were put into an ultrasonic bath for 3 min, shortly spun down, and then incubated overnight in a thermo shaker at 37°C. On the next day, tryptic proteolysis was inhibited by adding 20 µL of 100 mM phenylmethylsulfonylfluoride (PMSF). Preparation of PMSF is shown in the Appendix in Section 8.5.4. Samples were incubated for 2 h at RT. Samples were allowed to cool down to RT. After the inhibition of tryptic digestion, samples were transferred into 20 kDa filter. A 2 L beaker was completely filled with 25 mM NH4HCO3 and put on a magnetic stirrer. Twenty kDa filter containing samples were put in a floating tube rack and dialyzed for 24 h. After dialysis, samples were transferred into 1.5 mL tubes. Filter were washed twice with 50 µL 25 mM NH4HCO3 and transferred into the corresponding tube. Samples were dried in a centrifugal evaporator and stored at −20°C until the protocol was continued as described in Section 3.5.2. 39

Methods

3.4.2 Heat Mediated Antigen Retrieval The protocol for heat mediated antigen retrieval was mostly adapted from Hinneburg et al. (2017). Materials required for antigen retrieval are listed in Tab 12. Before starting Tris-HCl (0.1 M, pH 8.0) with 4% SDS, DTT (0.1 M), and urea (8 M) or NH4HCO3 (25 mM, pH 8.0) were prepared as described in the Appendix in Sections 8.5.2, 8.5.5, 8.5.6, and 8.5.7. One hundred microliter retrieval buffer consisting of 50 µL of Tris-HCl (0.1 M, pH 8) with 4% SDS and 50 µL of DTT (0.1 M) were added to each sample. Retrieval buffer was resuspended along the inside of the tube to ensure that all tissue sections were uptaken by the buffer. In case of microdissected tissue Section more preparation steps were required. Caps were hold upside down (Fig 27B) under a stereomicroscope (Fig 27D). Between 10 – 20 µL of retrieval buffer were added on the inside of the AdhesiveCap (Fig 27C), on which the microdissected tissues were attached. The buffer was carefully resuspended and then transferred into a new 1.5 mL tube. This step was repeated at least once until all microdissected tissues/cells were gathered (Fig 27F) and transferred into a new tube. The stereomicroscope is a useful device as sections are barely visible with the naked eyes. Next, tubes with microdissected tissue sections were filled with the remaining retrieval buffer to 100 µL. More than 100 µL in a 1.5 mL tube could result in unintended opening of the tubes during the following heating procedure. Furthermore, the capacity of the tube will not be sufficient to hold the volumes, which are necessary during the chloroform (CHCI3)-methanol precipitation (Hinneburg et al. 2017). All tissue sections were sonicated with a sonicator stick for ~ 15 s. The output power control was set to 7 – 8 out of 10. (Glyco)Proteins were denatured subsequently with mild agitation (600 rpm) at 99°C for 60 min in a thermo shaker. Afterwards, samples were allowed to cool down slowly to room temperature before they were centrifuged at ~ 2,000 g for 20 min. Supernatants (~ 100 µL) were transferred into new 1.5 mL tube. In the exact order 400 µL methanol, followed by 100 µL CHCI3 and 300 µL Milli-Q®-water were added and vigorously vortexed for 10 s. Samples were centrifuged again for 5 min at 14,000 g. Precipitated proteins were localized at the interphase (Fig 27G) between the methanol/water layer on the top and the CHCI3 layer at the bottom. The supernatant on the top was carefully removed and 300 µL methanol were very cautious added to the CHCI3 phase at the bottom. Tubes were vortexed for 10 s and then centrifuged for 5 min at ≥ 14,000 g. Protein pellets were spun down to the bottom (Fig 27H) and the entire supernatant was removed carefully. Microdissected tissue sections may not leave a noticeable pellet (Fig 27I). Therefore, supernatant was removed under the stereomicroscope. As pellets may detach from the bottom with time, it is not recommended to precipitate many (glycol)proteins at once. For N-glycan release from dot-blotted glycoproteins (Section 3.5.1), pellets were dissolved in 10 – 50 µL urea (8 M), depending on protein amount. Otherwise pellets were dissolved in 50 µL of 25 mM NH4HCO3 (pH 8.0) and the protocol continued in Section 3.5.2. Pellets were resolved via ultrasonic bath or by aspirating and dispensing the solution with a pipette. Samples were either stored at −20°C until usage or the protocol was continued accordingly.

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Methods

Fig 27: Preparational Steps for Heat Mediated Antigen Retrieval. A = Deparaffinized whole tissue in 1.5 mL tube; B = microdissected cells adhered on AdhesiveCap; C = buffer was added on the underside of the lid and D = visualized under a stereomicroscope; E = magnification and visualization of epithelial cells of roe deer epididymis; F = all cells transferred into a 1.5 mL tube; G = precipitated proteins in the interphase after second centrifugation step; H = protein pellet at the bottom after third centrifugation step; I = protein pellet of laser Microdissection samples are hardly visible. Supernatant should be removed very carefully, in certain circumstances under the stereomicroscope.

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3.5

N-Glycan Release, Purification and Derivatization

3.5.1 N-Glycan Release from Dot-Blotted Glycoproteins onto PVDF Membranes The dot-blotting procedure was mainly adapted from Jensen et al. (2012) and Hinneburg et al. (2017). Materials required for this section are listed in Tab 13. Before starting, four solution had to be prepared: stain solution (Section 8.5.8), destain solution I and II (Section 8.5.9), and polyvinylpyrrolidone 40 (PVP40) solution (Section 8.5.10). (Glyco)Proteins used for dotblotting should be dissolved in 8 M urea previously. According to the number of protein spots required, a suitable piece of polyvinylidene fluoride (PVDF) membrane was carefully cut. The inner lid of a 0.5 mL tube was pressed onto PVDF membrane serving as marked (glyco)protein spot area. Approximately ~ 1 cm space was set free between each spot. The intent of this engraved border was to simplify finding the protein spot after destaining the membrane. The PVDF membrane was activated by placing it on the top of a methanol-wetted lint free tissue (e. g., Kimtech wipe). Activating the membrane facilitates (glyco)protein capture. But no excess methanol should cover the membrane-surface before and while applying the samples. Otherwise the (glyco)protein solution would be washed away before (glyco)proteins are captured into the membrane. Each (glyco)protein solution was applied on individual spots with 2 µL at a time. As soon as the spots were dried, additional 2 µL were given until the desired (glyco)protein amount was accomplished. An appropriate amount of (glyco)proteins per spot are 5 – 20 µg for 45 µg thick PVDF membranes. The tissue underneath the membrane was kept profoundly wetted throughout the spotting procedure by applying methanol drops at the edges of the tissue periodically (~ 2 – 4 min). Finally, the PVDF membrane was air-dried at RT overnight to ensure proper binding of (glyco)proteins. The following wetting procedures in this paragraph were performed while the membrane was gently tilted on a gyratory shaker. The dried PVDF membrane was taken by a plastic tweezer and replaced into a glass petri dish. Methanol was filled into the petri dish and the membrane was rewet for 15 min. The membrane was turned over several times as it is hydrophobic. After 15 min, methanol was discarded. This procedure was repeated with Milli-Q®-water to remove unnecessary salts. Milli-Q®-water was discarded as well. Staining of (glyco)protein spots was performed using ~ 30 mL Coomassie® Brilliant Blue. Procedure was stopped as soon as (glyco)protein spots were stained appropriately. In a way, it can be stated that boldness of each stained spot represented their (glyco)protein amount. Stain solution was discarded and spots were destained in 30 mL destain solution I for 10 min. The procedure was repeated with destain solution II. Finally, the membrane was air-dried and could be stored for months at RT (Hinneburg et al. 2017). The protocol was continued by adding 100 µL PVP40 solution for each sample in a 96-well plate. Destained (glyco)proteins were cut with clean scissors or a scalpel along its engraving which was accomplished earlier by the inner lid of the 0.5 mL tube. Protein spots were put into the wells filled with the 100 µL PVP40 solution with the protein site facing up (Fig 28). From

42

Methods

this point, spots must not turn during the following steps. The plate was placed on the gyratory shaker and tilted mildly for 5 min before the PVP40 solution was removed. Spots were washed by repeating the previous procedure with Milli-Q®-water for three times. PNGase F solution of 5 µL per sample was prepared during the washing procedure. •

For PNGase F provided by New England Biolabs, ~ 125 U are needed which corresponds to 0.25 µL plus 4.75 µL of 25 mM NH4HCO3 (pH 8.0).

•

For PNGase F provided by Roche, ~ 0.25 – 0.5 U are needed which corresponds to 0.25 – 0.5 µL plus 4.5 − 4.75 µL of 25 mM NH4HCO3 (pH 8.0) (Johansen et al. 2009, Hinneburg et al. 2017).

Fig 28: Dot-Blotted Glycoproteins. Standard glycoproteins, transferrin top row and fetuin B bottom row, were not destained on purpose in order to demonstrate staining intensity according to glycoprotein amount. Left column: 5 µg glycoprotein; middle column: 10 µg glycoprotein; right column: 30 µg glycoprotein spotted.

After the washing procedure, 5 µL of PNGase F solution was added onto the (glyco)protein spots. The well plate was incubated for 10 min at 37°C, before additional 10 µL of 25 mM NH4HCO3 (pH 8.0) were added on each spot. Furthermore, 100 µL of water were added to all empty wells to avoid evaporation. The plate was sealed with a lid or by parafilm and then incubated at 37°C overnight in a heating oven. For MS approaches PNGase F should be obtained in glycerol-free version (Kolarich et al. 2015). On the next day, the 96-well plate was sonicated in ultrasonic bath for 5 min and then spun at 500 g for 1 min. Fluids at the bottom, containing N-glycans, were transferred into 1.5 mL tubes. Wells were washed twice with 15 µL Milli-Q®-water by resuspending along the inside an also transferred into the corresponding tube. Finally, an amount of ~ 55 µL Milli-Q®-water was added to achieve a final volume of 100 µL. Samples were then stored at −20°C until usage or the protocol was continued as described in Section 3.5.3. 43

Methods

3.5.2 N-Glycan Release in Solution N-glycans from denatured (glyco)proteins dissolved in 50 µL of 25 mM NH4HCO3 (pH 8.0) were also released directly in solution. Some steps, either preparational or operational, overlap in both approaches for N-glycan release. Materials needed for N-glycan release in solution are listed in Tab 14. PNGase F solution was prepared similar to the protocol in Section 3.5.1. While the amount of PNGase F per sample was the same, 25 mM NH4HCO3 (pH 8.0) was added to a final volume of 100 µL per sample. Thereafter, samples were sonicated for 3 min in an ultrasonic bath and shortly spun down. Tubes were put into a thermo shaker and incubated at 37°C overnight. Released N-glycans were stored at −20°C until usage. The protocol was continued in Section 3.5.3.

3.5.3 N-Glycan Purification N-glycan purification was based on RP-SPE as mentioned in Section 2.5.1. Materials used in this protocol are listed in Tab 15. The procedure was performed using Pierce® C18 Tips. Depending on sample amount to be purified, either a regular or a multichannel pipette was used. In the first case, the following solutions were transferred into 1.5 mL tubes. A multichannel pipette was employed to purify multiple samples at once. In this case, solutions were transferred into a 96-well plate as shown in Fig 29. Three types of solution were prepared: •

Wetting solutions: 200 µL methanol, 0.1% 400 µL TFA, 200 µL 40% ACN containing 0.1% TFA, and 80% ACN containing 0.1% TFA.

•

Equilibration solution: 1 mL Milli-Q®-water.

•

Rinse solution: 100 µL 0.1% TFA.

Independent of pipette type used, 100 µL was set according to the filling capacity of C18 tips. Throughout the purification procedure C18 tips must not run dry while changing solvents. Purification started with wetting C18 tips by slowly aspirating and dispensing (i) methanol, (ii) 0.1% TFA, (iii) 40% ACN containing 0.1% TFA, and (iv) 80% ACN containing 0.1%TFA. Each wetting solution was aspirated and dispensed twice (2 x 100 µL) and discarded afterwards. C18 tips were equilibrated with 1 mL Milli-Q®-water by applying the same procedure as for wetting solutions. Next, samples were slowly aspirated and dispensed 30 times. As hydrophile N-glycans were solved in aqueous phase, most N-glycans were not adsorbed by C18. Therefore, this solution must not be discarded. Remaining N-glycans were dissolved by aspirating and dispensing 0.1% TFA which was transferred to the sample solution. In case of applying a multichannel pipette and 96-well plate, samples in NH4HCO3 had to be transferred into a 1.5 mL tube. Finally, samples were dried in a centrifugal evaporator and then stored at −20°C until usage. The protocol was continued in Section 3.5.4, if HPLC separation was intended. For MS analysis only, samples were permethylated as described in Section 3.7.

44

Methods

Fig 29: N-Glycan Purification Using Multichannel Pipette and 96-Well Plate. Abbreviations: ACN = acetonitrile; NH4HCO3 = ammonium bicarbonate; TFA = trifluoroacetic acid. Note: Drawings (96-well plate, multichannel pipette, and 1.5 mL tube) were obtained from the internet. Corresponding inks are given in Section 7.1.

3.5.4 Derivatization with 2-Aminobenzamide This protocol for reductive amination with 2AB was mainly adapted from Bigge et al. (1995) and Wuhrer et al. (2009b). Materials used for this protocol were listed in Tab 16. Before starting, preparatory actions were conducted. This included warming up the thermo shaker to 65°C and preparing the 2AB solution as described in the Appendix in Section 8.5.11. If 2AB was already available, frozen label reagent was thawed by sonication for 3 min. Five microliter of 2AB label reagent was added per sample. To ensure, that all glycans were absorbed by the fluid, the label reagent was resuspended within the tube and sonicated in the ultrasonic bath for 3 min. Samples were spun down with a micro centrifuge for ~ 10 s. Reductive amination was achieved through incubation at 65°C for 3 h. Once an hour and at the end of incubation time, samples were sonicated for 3 min. 2AB labeled glycans had been allowed to cool down 45

Methods

to RT before they were dried in a centrifugal evaporator. Dried samples were stored at −20°C until usage. The protocol was continued in two possible ways. First, samples were directly prepared for HPLC analysis (Section 3.6) or second, purified from excessive label reagent (Section 3.5.5) and then analyzed by HPLC

3.5.5 Purification of N-Glycans from Excess 2-Aminobenzamide After derivatization with 2AB, N-glycan purification was performed with 100% cotton wool which were purchased in local stores. 100% cotton wool have two major advantages. First, acquisition cost is very low, and second, wax and proteins are removed during the manufacturing process. Hence, cotton wools are composed of pure cellulose (Selman et al. 2011). Materials needed for purification of excess 2AB are listed in Tab 17.

Fig 30: Preparation of 100% Cotton Wool served as HILIC SPE Microtip. A = An example of cotton wool obtainable from regular super markets; B = showing a size proportion of approximately 500 µg cotton wool; C = 500 µg cotton wool was pushed down into the end of a 10 µL tip by using a cannula; D = prepared HILIC SPE microtip. Abbreviations: HILIC = hydrophilic interaction liquid chromatography; SPE = solid phase extraction. Source: Selman et al. (2011), page 2494.

The following protocol was mainly adapted from Selman et al. (2011). An estimated amount of 500 µg cotton wool (Fig 30B) was pushed into the end of a 10 µL tip by using a cannula (Fig 30C). Prepared tips were used as a versatile HILIC-SPE tool. The cotton wool should not be pressed to hard into the tip as solvents would not be able to flow through any more. Functionality was tested by starting with the wetting solution. Non-functional HILIC-SPE tips were discarded immediately.

46

Methods

The purification procedure was also inspired by Selman et al. (2011). Similar to the first Nglycan purification step (Section 3.5.3) the protocol can be carried out with a regular pipette or by a multichannel pipette for treatment of multiple samples at once (Fig 31). Dried samples were dissolved in 90% ACN and sonicated for 3 min in ultrasonic bath. Next, four types of solution were prepared and transferred either into 0.5 mL tubes or into wells of a 96-well plate (Fig 31). •

Wetting solution: 50 µL Milli-Q®-water.

•

Conditioning solution: 30 µL 90% ACN.

•

Washing solution: 30 µL 90% ACN containing 0.1% TFA.

•

Elution solution: 5 µL Milli-Q®-water.

Fig 31: N-Glycan Purification of Excessive 2AB Using Multichannel Pipette and 96-WP. Wells were filled with 100 – 200 µL of each solution in order to facilitating pipetting (except samples). In contrast to the first N-glycan purification step (Section 3.5.3, Fig 29), up to 64 samples could be purified with a single 96-well plate Abbreviations: 2AB = 2-aminobenzamide; ACN = acetonitrile; HPLC = high performance liquid chromatography; TFA = trifluoroacetic acid; WP = well plate. Note: Drawings (96-well plate, multichannel pipette, and HPLC vial) were obtained from the internet. Corresponding inks are given in Section 7.1.

47

Methods

Prepared HILIC-SPE tips were washed five times with 10 µL of Milli-Q®-water and then conditioned by aspirating and dispensing 10 µL 90% ACN three times. Next, samples were slowly aspirated and dispensed 30 times in order to allow N-glycan adsorption. Afterwards, adsorbed N-glycans were washed three times using 10 µL of 90% ACN containing 0.1% TFA. Sample and washing solutions were transferred into individual 0.5 mL tubes and served for proof of principle. N-glycans were eluted with 5 µL Milli-Q®-water into HPLC vials. Lastly, samples were shortly dried in a centrifugal evaporator. Every HILIC microtip was used only once, although the microtips were used multiple times by Selman et al. (2011), as the costs are low and the microtips can easily be prepared. Samples were stored at −20°C until HPLC analysis (Section 3.6).

3.6

High Performance Liquid Chromatography

Three different columns were used and compared due to separation efficiency. The HPLC system used is shown in Fig 32. Materials used for HPLC analysis are listed in Tab 18.

Fig 32: HPLC System Used in this Work. A = HPLC system with column oven, autosampler, pump, and degasser; B = fluorescence detector; C = ZIC-HILIC column placed in column oven. Note: Manual injector, used solvents, computer and other electronical devices are not shown. Abbreviations: HILIC = hydrophilic interaction liquid chromatography; HPLC = high performance liquid chromatography; ZIC = zwitterionic.

48

Methods

3.6.1 Column I: TSKgel® Amide-80 Å, 4.6 mm x 25.0 cm The “TSKgel® Amide-80, 4.6 mm x 25.0 cm” was the first HILIC-column tested. The gradient separation was adapted from Wuhrer et al. (2009b). Solvent A consisted of 80% ACN and 20% 50 mM NH4HCO2 (pH 4.4). Solvent B was the reverse of solvent A, 20% ACN and 80% 50 mM NH4HCO2 (pH 4.4). The preparation of 50 mM NH4HCO2 is described in the Appendix (Section 8.5.12). Column temperature was adjusted to 40°C. The gradient separation used is shown in Tab 1. Total run time was 120 min. The flow rate was set to 1 mL min−1 to achieve the recommended column backpressure of 10 – 20 MPa. The HPLC system was set to a maximum of 40 MPa back pressure for precaution.

Tab 1: Gradient Separation with HILIC-Column I. Time Flow rate: 1 mL min−1

Solvent A 80% ACN, 20% 50 mM NH4HCO2

Solvent B 20% ACN, 80% 50 mM NH4HCO2

t = 0 min

100%

0%

t = 20 min

80%

20%

t = 90 min

40%

60%

t = 91 min (washing)

0%

100%

t = 110 min (washing)

0%

100%

t = 111 min (equilibration)

100%

0%

t = 120 min (end of run)

100%

0%

Abbreviations: ACN = acetonitrile; HILIC = hydrophilic interaction liquid chromatography; NH4HCO2 = ammonium formate.

Prior to sample application, the HPLC system was run for at least 15 min in order to degas and equilibrate the system. The fluorescence detector was set according to excitation (360 nm) and emission (425 nm) wavelength of 2AB (Wuhrer et al. 2009b). The sensitivity was adjusted to “5” (available sensitivities: 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 100, 1000). While the HPLC system was preparing, dried samples were dissolved in 100 µL solvent A. Samples were sonicated for 3 min in an ultrasonic bath and shortly spun down afterwards. The half was injected per HPLC run. Manual injection was performed by using a Hamilton syringe (50 µL). Before injection the syringe was cleansed by aspirating and dispensing five times 50 µL isopropanol, five times 50 µL Milli-Q®-water and another five times 50 µL Milli-Q®-water from a different Falcon tube. Samples were injected in Rheodyne 7125 at “Load” position. The size of sample loops utilized were between 100 − 200 µL. The handle was switched to “Inject” position and the HPLC run started with a manually initiated short. If automatic injection through autosampler was applied, all samples had to be transferred into HPLC glass vials. While samples purified from excessive 2AB (Section 3.5.5) were already located into the correct vials, samples not purified were previously air-dried in 1.5 mL tubes (Section 3.5.4). Cleaning prior to injection was carried out automatically by the autosampler with 80% isopropanol. Size of sample loop was the same as for Rheodyne 7125. Furthermore, 49

Methods

the autosampler was set to pause at least 2 min per samples. The personal computer was old and it could crash occasionally and the aim of the pause was to avoid trouble with the hardware.

3.6.2 Column II: SeQuant® ZIC®-HILIC, PEEK Coated, 200 Å, 150 x 4.6 mm The “SeQuant® ZIC®-HILIC, PEEK coated HPLC column, 3.5 µm, 200 Å, 150 x 4.6 mm” was the second HILIC-column tested. A total of 37 different gradient separation were tested until the optimum was determined. According to the manufacturer’s instruction, solvent A was 100% ACN and solvent B 5 mM NH4CH3CO2 (pH 6.4). The preparation of 5 mM NH4CH3CO2 (pH 6.4) is described in the Appendix (Section 8.5.13). Column temperature was adjusted to 40°C. The best gradient separation determined is shown in Tab 2. Total run time was 105 min. The flow rate was set to 200 µL min−1 to achieve the recommended column backpressure of 10 – 20 MPa. The HPLC system was set to a maximum of 40 MPa back pressure as reason for precaution.

Tab 2: Gradient Separation with HILIC-Column II. Time Flow rate: 200 µL min−1

Solvent A 100% ACN

Solvent B 5 mM NH4CH3CO2

t = 0 min

75%

25%

t = 5 min

65%

35%

t = 60 min

40%

60%

t = 65 min (washing)

0%

100%

t = 75 min (washing)

0%

100%

t = 80 min (washing)

100%

0%

t = 80 min (washing)

100%

0%

t = 91 min (equilibration)

75%

25%

t = 105 min (end of run)

75%

25%

Abbreviations: ACN = acetonitrile; HILIC = hydrophilic interaction liquid chromatography; NH4CH3CO2 = ammonium acetate.

Preparation of HPLC and setting the fluorescence detector were performed as described previously (Section 3.6.1). Dried samples were dissolved in 20 µL (75% solvent A, 25% solvent B), sonicated for 3 min in an ultrasonic bath and shortly spun down afterwards. The half was injected per HPLC run. Manual injection differed from the previous approach (Section 3.6.1). A 25 µL Hamilton syringe was used and therefore, a less amount of isopropanol and Milli-Q®-water used for cleaning. Samples were injected in Rheodyne 8125 at “Load” position. The size of sample loops utilized were between 25 − 50 µL. In contrast to Rheodyne 7125, version 8125 started the HPLC run automatically after switching the handle to the “Inject” position.

Sample application via

autosampler was comparable to the first column approach (Section 3.6.1). Only run time and sample loops were adapted accordingly. 50

Methods

3.6.3 Column III: SeQuant® ZIC®-HILIC, PEEK Coated, 100 Å, 250 x 2.1 mm The “SeQuant® ZIC®-HILIC, PEEK coated HPLC column, 3.5 µm, 100 Å, 250 x 2.1 mm” was the last HILIC-column tested. In contrast to the former column, a pre-column was used in this approach. Only five different gradient separations were tested until the optimum was determined as the column is comparable to the previous one. Solvents, column temperature and back pressure were not changed (Section 3.6.2). The best gradient separation determined is shown in Tab 3. Total run time was 170 min. The flow rate was set to 100 µL min−1.

Tab 3: Gradient Separation with HILIC-Column III. Time Flow rate: 100 µL min−1

Solvent A 100% ACN

Solvent B 5 mM NH4CH3CO2

t = 0 min

75%

25%

t = 10 min

65%

35%

t = 120 min

40%

60%

t = 125 min (washing)

0%

100%

t = 135 min (washing)

0%

100%

t = 140 min (washing)

100%

0%

t = 150 min (washing)

100%

0%

t = 151 min (equilibration)

75%

25%

t = 170 min (end of run)

75%

25%

Abbreviations: ACN = acetonitrile; HILIC = hydrophilic interaction liquid chromatography; NH4CH3CO2 = ammonium acetate.

All other preparation steps and manual injection procedure did not differ from the second approach (Section 3.6.2). Sample application via autosampler was comparable to the first column approach (Section 3.6.1). Only run time and sample loops were adapted accordingly.

3.7

Permethylation

All samples were evaporated prior permethylation as sodium hydroxide (NaOH) is hygroscopic and the absorbance of water would interfere with the reaction (Zhou et al. 2017). All glass vials were heated at 500°C for 5 h to avoid contamination. Materials used for permethylation of Nglycans are listed in Tab 19. The permethylation protocol was additionally adapted with ideas from Berman et al. (1981) and Morelle and Michalski (2007). All samples were incubated for 24 h in an exsiccator before starting. Whenever possible, permethylation was performed under a fume hood as toxic solvents were used. Furthermore, all steps involving NaOH, DMSO, and methyl iodide (ICH3) were conducted under argon atmosphere. Three NaOH per sample were placed in a dry mortar and transformed into fine powder through crushing by a pestle. Approximately 25 µg NaOH were put into a dry glass vial. A septum silicon (16 mm) was placed inside the cap. Aggressive solvents like dichloromethane (CH2Cl2) may dissolve organic components through contact which could 51

Methods

increase the baseline signal (noise). Dried samples were dissolved in 300 µL ultra-pure and dry DMSO and sonicated for 3 min in an ultra-sonic bath. After that, dissolved samples were transferred into glass vials containing NaOH. Non-reduced N-glycans were vigorously vortexed for 1 min while reduced N-glycans needed 5 min. Vortexing of multiple samples at once was achieved by bonding a vial holder on a thermo shaker at RT. The highest possible agitation (~ 1,400 rpm) was set. Samples were shortly (~ 10 s) centrifuged. 50 µL ICH3 were added per sample and glass vial was flushed with a stream of argon before closing the lid. Non-reduced N-glycans were vigorously vortexed for 30 min (reduced N-glycans 40 min) as outlined previously. After that, samples were shortly spun down. Following steps are performed at 4°C by placing glass vials in a Styrofoam box filled with crushed ice. Methylation reaction was stopped by adding 1 mL 10% acetic acid. Samples were shortly vortexed (~ 10 s). Next, 1 mL of CH2Cl2 was added. CH2Cl2 extraction ensured recovery of permethylated N-glycans (Berman et al. 1981). Non-reduced N-glycans as well as reduced N-glycans were vigorously vortexed for 5 min and shortly spun down as outlined previously. The CH2Cl2 layer containing N-glycans was on the bottom and the upper layer was removed. 1 mL 10% acetic acid was added to each sample and the procedure was repeated once more. Thereafter, 1 mL Milli-Q®-water was added and samples were vigorously vortexed for 10 s and shortly centrifuged accordingly. The upper (water) layer was discarded. This step was repeated three to five times. Samples were incubated at −20°C until remaining water was frozen (approximately 30 min). The unfrozen CH2CI2 phase was quickly transferred into new glass vials and dried in a centrifugal evaporator. Dried permethylated N-glycans were either purified as shown in the Appendix (Section 8.4.1) or dissolved in 50 µL methanol and transferred into HPLC glass vials. Samples were stored at −20°C until MS analysis (Section 3.8).

3.8

Mass Spectrometry

Due to time issues, only MALDI-TOF-MS of permethylated (not 2AB labeled) N-glycans was performed. Tab 20 lists all materials needed for MALDI-TOF-MS. 2,5-DHB matrix was prepared by dissolving 10 mg 2,5-DHB in 1 mL 50% ACN (Yin et al. 2013) in a 1.5 mL tube. Matrix was sonicated for 3 min in an ultrasonic bath and stored at −20°C until usage. One microliter of sample solution (in methanol) was spotted on a stainless steel AnchorChip target and immediately mixed with 1 µL of 2,5-DHB matrix by resuspending. Drops were airdried at RT allowing crystals to form (Zaia 2008). After transferring all samples onto the target, AnchorChip was put into the MALDI-MS. The MS was controlled via flexControl installed on a personal computer. Mass spectra were recorded in positive-ion mode (Kang et al. 2005) over the range 500 – 5,000 m/z. Twenty-five shots were accumulated until an appropriate mass spectra were achieved (125 – 250 shots in total needed). Each mass spectra were saved and analyzed with flexAnalysis and GlycoWorkbench. After all spots (384 in total) were used, the AnchorChip was cleaned according to Signor and Boeri Erba (2013). The cleaning protocol of the AnchorChip is described in the Appendix (Section 8.4.2).

52

Results

4

Results

4.1

Establishment of an HPLC Protocol

4.1.1 Dextran Ladder Starting with the HILIC column TSKgel® Amide-80 Å, two additional HILIC columns were obtained in the process of time and proved due to their separation efficiency. The establishment of a HPLC protocol started with 2AB labeled dextran. For each run, 9.09 µg dextran were injected into the HPLC system. Pure Milli-Q®-water or 25 mM NH4HCO3 with 2AB label were used as blanks. Separation efficiency of dextran was tested ten to sixty times per column since a suitable gradient was still to be established for column II (Section 3.6.2) and column III (Section 3.6.3). The expected dextran ladder was achieved by all columns (Fig 33). Nevertheless, significant differences in separation efficiency were observed.

Fig 33: Dextran Ladder Separated by Three Different Columns. A represents dextran separation with column I while B shows the following rinsing after the dextran run with Milli-Q®-water. C and D represent the same for column II and E and F for column III.

53

Results

Peak tailing and shoulder peaks were only observed at the beginning in column I, most likely caused through aging of the analytical column. After removing air bubbles from the HPLC pump, checking the functionality of the degasser, closing all tubes leakproof, and rinsing the column appropriately no unacceptable asymmetric peaks were observed anymore. The left runs (separated graphs) of Fig 33 represents dextran separation from all HILIC columns. Both SeQuant® ZIC®-HILIC columns had sharper peaks and their digital chromatogram displays higher fluorescence intensity. The last column (Fig 33E) represented the best results since it porosity and diameter is the smallest. All systems were run with individual flow rates and gradient separation. The right runs (separated graphs) of Fig 33 shows a subsequent run with Milli-Q®-water. While column I (Fig 33B) and column II (Fig 33D) showed still fluorescent signals, column III (Fig 33F) had none. Taken together, column III delivered the highest separation efficiency and did not have to be rinsed with Milli-Q®-water between each run.

4.1.2 Standard Glycoproteins: Trypsin Mediated Antigen Retrieval The experiments continued with HPLC analysis of 2AB labeled N-glycans of standard glycoproteins (Sections 2.2.2, 2.2.3, and 2.2.4). Samples were treated as described in Sections 3.4.1, 3.5.2, 3.5.3, and 3.5.4. However, the initial RP-SPE purification of N-glycans with C18 tips used 400 µL of TFA as elution solution. After analyzing each 100 µL of eluted TFA, it was pointed out that only 100 µL was needed to elute all N-glycans (data not shown). The half of each sample was injected into the HPLC system (N-glycan amount of 100 µg glycoprotein). Blanks used as negative control were either Milli-Q®-water, 25 mM NH4HCO3 with 2AB label or any standard glycoprotein without PNGase digestion. No peaks were detected in any version (data not shown). Standard glycoproteins were analyzed at least five times. At that time, no purification of excessive label reagent was considered since N-glycan peaks were not interfered by free 2AB. Furthermore, the 2AB peak at the very beginning could be distinguished clearly to the N-glycan peaks. Fig 34A shows N-glycans of asialofetuin separated by column I. Since column I showed the weakest results, column I was replaced by column II. Much higher separation efficiency of asialofetuin N-glycans was demonstrated by column II (Fig 34B) and column III (Fig 34C), respectively, with the best in column III. Fig 34D and Fig 34E represent N-glycans of fetuin analyzed with column II and column III, respectively. Both chromatograms are shown without zooming to demonstrate the first major 2AB peak. It was convenient to spin down samples for 5 min at highest speed possible after dissolving them into the corresponding HPLC solvent. A visible pellet (presumably 2AB salts) was spun down and the supernatant was transferred to a new HPLC glass vial. This led to a smaller 2AB peak at the beginning as demonstrated in Fig 34E compared to Fig 34D. High-mannose glycans of RNase B are shown in Fig 34F. Although N-glycans of all glycoprotein standards are mostly known, no expected N-glycans were plotted in Fig 34 as it would be only an assumption. For proper assignment of N-glycans to HPLC peaks a subsequent MS analysis should be performed. However, MS analysis (Section 4.5) was carried out separately. 54

Results

Fig 34: 2AB Labeled N-Glycan Peaks of Tryptic Digested Standard Glycoproteins. Chromatogram A shows N-glycan peaks of asialofetuin separated by column I. B and C represent Nglycan peaks of asialofetuin by column II and column III, respectively. D (column II) and E (column III) show the full chromatogram of N-glycans of fetuin as well as a selected area of N-glycans. F shows Nglycans of RNase B separated by column II. Abbreviations: 2AB = 2-aminobenzamide; RNase = ribonuclease.

4.1.3 Standard Glycoproteins: Heat Mediated Antigen Retrieval N-glycan analysis of standard glycoproteins were also performed after a heat mediated antigen retrieval as described in Sections 3.4.2, 3.5.1 or 3.5.2, 3.5.3, and 3.5.4. It should be noted that the heat mediated antigen retrieval was utilized at a later time when only column III was used. Fig 35 shows 2AB labeled N-glycan peaks of human transferrin. The upper chromatograms (Fig 35A and Fig 35B) were treated as described previously (trypsin mediated antigen retrieval). N-glycans were released from 200 µg human transferrin. The heat mediated antigen retrieval was followed by dot-blotting glycoproteins on a PVDF membrane (Fig 35C and Fig 35D) or N-glycans were released directly, skipping the dot-blotting procedure (Fig 35E and Fig 35F). Unlike the first approach, only 25 µg of glycoprotein were spotted onto the PVDF 55

Results

membrane since it cannot be loaded with a higher protein amount per spot. Therefore, the heat mediated approach without dot-blotting was performed with the same amount of glycoprotein in order to compare the peak efficiencies. Peak areas were calculated by using EuroChrom for Windows and ImageJ. Although both areas were comparable, the noise of 2AB is slowly but steadily interfering N-glycan peaks at lower concentrations (Fig 35D and Fig 35F). The eightfold amount of N-glycans in the first approach (Fig 35A and Fig 35B) is the reason why the peak intensities were much higher. Besides more peaks were detected in the first approach.

Fig 35: 2AB Labeled N-Glycan Peaks of Human Transferrin. Chromatograms A and B show N-glycan peaks of transferrin as it was described for fetuin (Fig 34). The lower four chromatograms represent N-glycans of transferrin after a heat mediated process. Nglycan release of samples represented in C and D were performed without prior dot-blotting on a PVDF membrane. E and F on the other hand, were dot blotted prior to N-glycan release. Note: left columns were in original size while right columns were zoomed appropriately. Abbreviations: 2AB = 2-aminobenzamide; PVDF = polyvinylidene fluoride.

56

Results

4.1.4 Limit of Detection Laser microdissected samples will most likely not contain a huge amount of N-glycans as it was demonstrated with standard glycoproteins. For that, limit of detection (LOD) and therefore the usefulness of the system was defined before FFPE tissue sections and laser microdissected samples were analyzed. LOD was performed with both, column II and column III, as well as both antigen retrieval approaches. Each glycoprotein amount was diluted 1:1 beginning with 200 µg until ~ 0.195 µg. Dilution was performed after N-glycan purification and 2AB labeling was completed. This has two major advantages. First, less samples have to be prepared at once and second, costs could be saved as PNGase F and C18 tips are expensive. The half amount was injected per HPLC run as in previous experiments. Starting with the results of column II, Fig 36 represents some chosen chromatograms of different amounts of released N-glycans from RNase B. Antigens were retrieved through tryptic digestion. The magnification of all chromatograms was put on the same level to ensure that peak intensities can be compared. Other than expected, the peaks of minor N-glycan amounts (Fig 36C and Fig 36D) showed higher peak intensity and did not fall proportionally to the concentration.

Fig 36: LOD for 2AB Labeled N-Glycans of RNase B Separated with Column II. Amount of N-glycans released from A = ~ 25 µg glycoprotein; B = ~ 12.5 µg glycoprotein; C = ~ 0.7813 µg glycoprotein; D = ~ 0.0977 µg. Abbreviations: 2AB = 2-aminobenzamide; LOD = limit of detection; RNase = ribonuclease.

RNase B has a single glycosylation site at Asn34 (Bernard et al. 1983) as mentioned earlier (Section 2.2.3). Given the identified N-glycans published by Fu et al. (1994) and Prien et al.

57

Results

(2009) shown in Tab 24 the mean molecular mass of N-glycans is ~ 1,500 Da. With a total molecular mass of ~ 15,500 Da (Bernard et al. 1983) and assuming that all high-mannose glycans appear equally, the N-glycan mass percentage is ~ 9.9%. Since released N-glycans from 0.0977 µg RNase B were still detected by the system the LOD is at least 6.29 pmol. LOD was also analyzed with column III (Fig 37). Sample treatment was similar to the previous approach. Column III sill delivered better N-glycan separation and sharper peaks. In comparison with column II the LOD was identical, however, all areas did decrease proportionally.

Fig 37: LOD for 2AB Labeled N-Glycans of RNase B Separated with Column III. Amount of N-glycans released from A = ~ 50 µg glycoprotein; B = ~25 µg glycoprotein; C = ~ 12.5 µg glycoprotein; D = ~ 1.5625 µg. Abbreviations: 2AB = 2-aminobenzamide; LOD = limit of detection; RNase = ribonuclease.

Lastly, it was tested if areas also decrease proportionally and/or antigens were retrieved through a heat mediated process. Antigens from 100 µg and 50 µg of human transferrin were retrieved through heat mediated process and N-glycans were released in solution (without dotblotting). N-glycans were 2AB labeled, purified and half of the amount were injected into the HPLC system (Fig 38). All areas were calculated and decreased proportionally with falling concentration. LOD was not tested since no other results were estimated for this approach yet. Despite the high amount of N-glycans analyzed (Fig 38A) no more peaks were detected as it was shown for N-glycans which were released from tryptic digested human transferrin (Fig 35B).

58

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Fig 38: LOD for 2AB Labeled N-Glycans of TF after Heat Mediated Antigen Retrieval. Amount of N-glycans released from A = ~ 50 µg glycoprotein; B = ~ 25 µg glycoprotein. Abbreviations: 2AB = 2-aminobenzamide; LOD = limit of detection; TF = transferrin.

4.2

N-Glycan Analysis of Whole FFPE Tissue Sections

Unlike standard glycoproteins for which it did not matter what antigen retrieval approach was utilized, no signals were detected for whole FFPE tissue sections after tryptic digestion (data not shown). Multiple approaches of tryptic digestion were tested as described down below. Beside the regular methods described in Sections 3.2.2 and 3.2.3 two other approaches were additionally tested. Trypsin was directly applied on the tissue section and incubated for 24 h in a humidity chamber. The liquid supernatants were transferred into a 1.5 mL tube. Histology glass slides were washed twice with 20 µL 25 mM NH4HCO3 and the liquid was also transferred into the same tube. The protocol was continued accordingly with dialysis (Section 3.4.1). Another approach was to apply PNGase F directly onto histology glass slides without antigen retrieval. No matter which concept was tested, every experiment utilizing none or tryptic digestion has failed. That was the reason why heat mediated antigen retrieval (Jensen et al. 2012, Hinneburg et al. 2017) was tested in the first place (Sections 4.1.3 and 4.1.4). With this approach N-glycans were detected regardless of deparaffinization method or using dot-blotting procedure. The Reproductive Biochemistry Unit at the Institute of Reproductive Biology (Leibniz Institute of Farm Animal Biology) was investigating Fzt:DU mice and the opportunity was taken at the very moment to obtain organs they did not require, like epididymis or testis. Fzt:DU are an outbred mouse strain (Bunger et al. 1998) which were already tested as control to high-fertility mouse lines like FL1 (Spitschak et al. 2007). Therefore, samples were freshly FFPE prior to the analysis. Fig 39 represents results from N-glycan analysis of whole FFPE tissue sections. The upper four chromatograms (Fig 39A, Fig 39B, Fig 39C, and Fig 39D) represent 2AB labeled Nglycans of the epididymis from a Fzt:DU mouse. The lower two (Fig 39E and Fig 39F) displayed chromatograms of the testis from the same mouse. The area of the analyzed epididymis was approximately 150 mm2 and testis nearly two-third (~ 90 mm2). Four different approaches were tested for the epididymis. The first two samples, Fig 39A and Fig 39B, were deparaffinized and scratched off accordingly (Section 3.2.2) while the next two, Fig 39C and Fig 39D, were deparaffinized in 1.5 mL regular or LoBind tubes (Section 3.2.3). The first sample (Fig 39A) 59

Results

was incubated with PNGase F in 25 mM NH4HCO3 (Section 3.5.2) while the second sample (Fig 39B) was dot-blotted on a PVDF membrane prior to PNGase F digestion (Section 3.5.1).

Fig 39: Analysis of 2AB Labeled N-Glycans from Whole FFPE Tissue Sections. Chromatogram A represents N-glycans of the epididymis obtained from Fzt:DU mouse. After heat mediated antigen retrieval, N-glycans were released directly without dot-blotting of glycoproteins. Chromatogram B represents the same approach but N-glycans were released from dot-blotted glycoproteins. Chromatograms C and D were deparaffinized in normal or LoBind 1.5 mL tubes, respectively. In both cases N-glycans were released directly in 25 mM NH4HCO3 (like A) without dotblotting any glycoproteins. Chromatograms E and F represents N-glycans of testis obtained from Fzt:DU mouse. Both tissue sections were scratched of the histology glass slide but only approach F used dot-blotting of glycoproteins prior N-glycan release. Abbreviations: 2AB = 2-aminobenzamide; FFPE = formalin-fixed paraffin embedded; NH4HCO3 = ammonium bicarbonate.

The best result was found in the first attempt. Unlike the standard glycoproteins (Fig 35D and Fig 35F), sharper peaks were observed when dot-blotting was renounced. Consequently, samples deparaffinized in tubes skipped the dot-blotting procedure. Samples deparaffinized and treated in regular 1.5 mL tubes (Fig 39C) showed comparable peaks to the first attempt 60

Results

(Fig 39A). N-glycan peaks were barely identified when everything was performed in 1.5 mL LoBind tubes. Similar observations were made for the testis sections (Fig 39E and Fig 39F). As expected, for both organs different peaks and retention times were observed. The testis showed lower peak intensity, however, it was comprehensible since the tissue area was much smaller. Regardless of the FFPE organic tissue or sample preparation, the noise presumably caused by excessive 2AB label interfered the chromatogram. Blanks used in this part of the experiments were single Paraplast® pellets deparaffinized in 1.5 mL regular or LoBind tubes. No significant peaks were observed (data not shown).

4.3

N-Glycan Analysis of Laser Microdissection Samples

Taken the results from Section 4.2 into account, laser microdissected cells were analyzed with the best method observed so far. Briefly, antigen retrieval was achieved through a heat mediated process (Section 3.4.2), followed by N-glycan release without dot-blotting (Section 3.5.2), N-glycan purification (Section 3.5.3), 2AB labeling (Section 3.5.4), and finally, preparing samples for HPLC analysis accordingly (Section 3.6.3). Negative controls were laser microdissected PET membrane regions in an equivalent area as laser microdissected cells. No peaks for the blanks were observed as shown in Fig 43C.

Fig 40: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Adult Mouse). A shows selected epithelial cells from Ductus epididymis in 5-fold magnification while B displays the same sector in 10-fold magnification. C and D are showing the corresponding chromatogram. Through to excessive label reagent, small peaks are barely visible. Note: Tissue section was stained with H&E. Abbreviations: H&E = Hemalaun and eosin.

61

Results

Results from laser microdissected epithelial cells from Ductus epididymis (adult Fzt:DU mouse) are shown first (Fig 40). The whole epididymis was analyzed as whole tissue sections previously (Section 4.2). Before microdissecting the epithelial cells, sperms located in the lumen were microdissected first. Therefore, it was possible to analyze both cell types. Furthermore, it simplified the cutting procedure for epithelial cells when sperms were already removed. Five complete areas were dissected with an area of 5.3 mm2 corresponding to ~ 1060 cells. Consequently, released N-glycans of ~ 530 cells were analyzed per HPLC run. Selected cells are shown in Fig 40A (5-fold magnification) and Fig 40B (10-fold magnification). H&E darken the sight, leading the LCM device to assign higher backlight and therefore, slowing down the system. N-glycan peaks were totally suppressed through excessive 2AB label reagent. As the N-glycan amount of laser microdissected cells are extremely low, N-glycan peaks were not able to permeate against the high signal intensity from the label reagent (Fig 40C and Fig 40D).

Fig 41: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Adult Rat). A shows selected epithelial cells from Ductus epididymis of an adult rat in 10-fold magnification while B displays an overview of dissected cells in 5-fold magnification. C shows microdissected cells adhered to the AdhesiveCap in 5-fold magnification and D the corresponding chromatogram. 2AB labeled N-glycan peaks were due to excessive label reagent not visible. Abbreviations: 2AB = 2-aminobenzamide.

Next, the same cell type from another organism (adult rat) was laser microdissected. Thirteen complete areas of epithelial cells from Ductus epididymis were dissected with an area of 12.30 mm2 and ~ 2,460 cells. N-glycans of ~ 1,230 cells were analyzed per HPLC run. After detaching sperms, complete areas of epithelial cells from Ductus epididymis were selected (Fig 41A) and microdissected (Fig 41B). This sample was stained only with Hemalaun leading 62

Results

to improved visibility compared to H&E. All gathered areas were adhered to the AdhesiveCap as it is shown in 5-fold magnification in Fig 41C. Except from the very beginning (zoomed chromatogram in Fig 40D) only jagged peaks were observed. It was not possible to determine peaks accordingly and therefore, peaks cannot readily be considered as N-glycans. Epithelial cells of Ductus epididymis were also analyzed from a third organism. A wildtype roe deer, deceased in August 2001, was stored in the archives of the Glycobiology Group at the Institute for Reproductive Biology (Leibniz Institute for Farm Animal Biology). The same applies to all other roe deer samples. All FFPE epididymis’s of roe deers were subdivided in caput, corpus, and cauda. As described for the previous samples, sperms in the lumen of the epithelial cells were removed beforehand. In all three cases, sperms were analyzed previously at a time in which trypsin mediated antigen retrieval was still used. Similar to the whole FFPE tissue sections, no N-glycan peaks were observed. Laser microdissected sperms are shown in Fig 50. The roe deer epididymis was stained with Hemalaun only as shown in Fig 42A and Fig 42B. A total of 16 complete epithelial areas from Ductus epididymis were microdissected. The obtained area of tissue section was 21.5 mm2 with ~ 4,300 cells, and therefore, N-glycans of ~ 2,150 cells for each HPLC run. An insight of adhered epithelial cells is shown in Fig 42C. As more cells were obtained this time, N-glycan peaks were slightly able to prevail against the noise caused by excessive label reagent. N-glycan peaks were still too strongly suppressed (Fig 42D). The second HPLC run as well as a new laser microdissected sample showed similar results. LCM was utilized for numerous other samples. For example, Fig 43A represents the chromatogram of sperms microdissected from the tail (cauda) of Ductus epididymis. The donor was a wildtype roe deer deceased in April 2001. Seven complete lumen containing sperms were microdissected. Even if the obtained area was small (6.93 mm2), approximately 3.500 cells were detached in total since about 500 sperms are located in a single lumen with an area of 1 mm2. The chromatogram of the next sample (Fig 43B) represents sperms microdissected from the tail (cauda) of Ductus epididymis as well. This time the wildtype roe deer was deceased in August 2001. Seven complete lumen containing sperms were microdissected as well. The obtained area was slightly bigger (7.56 mm2) and therefore, probably a few hundred more cells were detached. Although the retention time of the peaks differs, proper analysis of differences in Nglycosylation regarding the mating time was not possible.

63

Results

Fig 42: Laser Capture Microdissected Epithelial Cells from Ductus Epididymis (Roe Deer Deceased in August 2001). A shows selected epithelial cells from Ductus epididymis in 10-fold magnification while B displays an overview of dissected cells in 5-fold magnification. C shows microdissected cells adhered to the AdhesiveCap in 10-fold magnification and D the corresponding chromatogram. Through to excessive label reagent, small peaks are relatively visible.

64

Results

Fig 43: Laser Capture Microdissected Cells of Epithelial Cells from Ductus Epididymis. A shows the chromatogram of sperms located in the tail (cauda) of Ductus epididymis from a roe deer deceased in April 2001. B shows the chromatogram of sperms located in the tail (cauda) of Ductus epididymis from a roe deer deceased in August 2001. C shows the chromatogram of a membrane blank (20 mm2) with no significant peaks.

65

Results

4.4

Purification of Excess 2-Aminobenzamide Label Reagent

As N-glycan peaks of microdissected cells are suppressed through excessive 2AB label reagent, making the distinction of peaks impossible, purification steps were considered at the end of this work.

4.4.1 Hydrophilic Liquid Interaction Chromatography Solid Phase Extraction Purification through HILIC micro SPE (Section 2.5.3) was performed as described in Section 3.5.5. After 2AB labeling, N-glycans were dried using a centrifugal evaporator. DMSO is barely volatile. Disregarding the application time of the centrifugal evaporator the samples could not be recovered. A film of greasy DMSO was still left and could not be removed. Nevertheless, purification was tried anyway as shown in Fig 44. First, 200 µg asialofetuin was processed (the half amount was injected per HPLC run) as described previously (Section 4.1.3). After evaporating the sample (as far as it was possible), 2AB labeled N-glycans were purified with HILIC-SPE tips. Fig 44A represents the sample solution after aspirating and dispensing several times (Section 3.5.5). If N-glycans were adsorbed by the cotton wool, no N-glycan peaks should not be detected. The opposite was the case.

Fig 44: Purification of 2AB Labeled N-Glycans with HILIC-SPE. A represents the sample solution containing peaks of 2AB labeled N-glycans from 100 µg fetuin which should not be the case. The same goes for D which contained the half amount. B shows 2AB labeled N-glycan peaks of fetuin detected from the washing solution (should also not be the case). And C represents 2AB labeled N-glycan peaks obtained from the elution solution which should show the highest peak efficiency. Abbreviations: 2AB = 2-aminobenzamide; HILIC = hydrophilic interaction liquid chromatography; SPE = solid phase extraction.

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Results

This experiment was repeated with 100 µg of fetuin (N-glycans from 50 µg glycoprotein were injected per HPLC run). Fig 44B represents the washing solution which actually should not contain any peaks of 2AB labeled N-glycans. The same applies to the sample solution (Fig 44D). However, the peak area did fall proportionally. Fig 44C, the elution solution, should represent the full extent of all N-glycan peaks which was also not the case. Therefore, 2AB purification through HILIC-SPE tips was not successful.

4.4.2 Isocratic Separation Prior Gradient Separation At an earlier point of experiments, various gradient separation methods were tested to establish the HPLC protocol using ZIC®-HILIC columns with NH4CH3CO2 as solvent B. Some settings used an isocratic separation at the very beginning (Tab 4). As no advantages were observed at that time, partially isocratic separation was renounced.

Tab 4: Use of Isocratic Separation at the Beginning to Purify N-Glycans from Excessive 2AB Label (HILIC-Column II). Time Flow rate: 100 µL min−1

Solvent A 100% ACN

Solvent B 5 mM NH4CH3CO2

t = 0 min (isocratic)

98%

2%

t = 5 min

98%

2%

t = 10 min

75%

25%

t = 70 min

40%

60%

t = 80 min (washing)

0%

100%

t = 95 min (washing)

%

100%

t = 105 min (equilibration)

98%

2%

t = 120 min (end of run)

98%

2%

Abbreviations: ACN = acetonitrile; HILIC = hydrophilic interaction liquid chromatography; NH4CH3CO2 = ammonium acetate.

Due to the fact that laser microdissected samples cannot be analyzed with the high amount of fluorescence label, the isocratic separation at the beginning shall be reconsidered. This could serve as an online purification step. As shown in Fig 45 a major gap is observed between the 2AB label peak and the first dextran peak. This might occur with microdissected samples or whole FFPE tissue sections, respectively.

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3 Fig 45: Purification of Excessive 2AB Label Through Isocratic Separation. Abbreviations: 2AB = 2-aminobenzamide.

4.5

Mass Spectrometry Analysis of Permethylated N-Glycans

MALDI-MS analysis of permethylated N-glycans was performed at the very beginning as described in Sections 3.5.2, 3.7, and 3.8. No antigen retrieval was performed at this time but it could possibly improve the mass spectra. MALDI-MS analysis was used to establish the permethylation protocol. Neither MSn nor exoglycosidase digestion were performed and therefore, mass spectras showing in Fig 46 do not represent any fragmentations. Only N-glycan structures identified by Aich et al. (2013) were taken into account. N-glycans of both glycoproteins showed high signal intensities at 1,197 m/z, 1,533 m/z, and 1,713 m/z which could not be matched to an appropriate N-glycan structure. It is assumed that these m/z corresponds to B/Y fragmentations which was confirmed using GlycoWorkbench but not proven accordingly through MSn. Masses were calculated as [M + Na]+. The only regarded SiaAc were Neu5Ac and Neu5Gc in case of fetuin B. Calculated m/z and observed m/z differed slightly from 0 – 1.5% and therefore, MS analysis of permethylated N-glycans was successful. Every major peak identified by flexAnalysis 3.4 corresponded to an already identified N-glycan of asialofetuin or fetuin B, respectively (Aich et al. 2013). Nevertheless, more peaks were actually expected for both glycoproteins. Permethylation was successful as peak gaps occur exactly in 14 Da steps representing undermethylated, permethylated and over-methylated N-glycans.

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Results

Fig 46: Mass Spectrometry Analysis of Permethylated N-Glycans. Mass spectra A (blue) represents permethylated N-glycans of asialofetuin (laser intensity 20−30%; 225 shots taken); Mass spectra B (red) represents permethylated glycans of fetuin B (laser intensity 20 – 30%; 250 shots taken). Note: y-axis displays the intensity; x-axis displays m/z which was compared to the published results from Aich et al. (2013). Abbreviations: m/z = mass-to-charge ratio.

69

Discussion

5

Discussion

5.1

Establishment of an HPLC Protocol

LCM enables the procurement of a microscopic and contamination-free cellular sections away from its heterogenous tissue milieu (Lehmann et al. 2000, Liotta and Petricoin 2000, Liu 2010, Vandewoestyne et al. 2012, Hinneburg et al. 2017). Laser microdissected sample amounts are usually low. In order to achieve (optimal) results, protocols of bioanalytical devices have to be established first. Starting with the HPLC system three different columns were tested. The first column showed poor results. It fluorescent intensity, peak efficiency, and peak shape quality was too insufficient for LCM analysis. Furthermore, with its flowrate of 1 mL min−1 it caused high solvent consumption. Solvents used by column I were ACN and NH4HCO2. The latter favored the formation of charged ions, resulting in fragmentation of O-glycosidic linkages which leads to less giving informative MSn spectra (Kolarich et al. 2015). Consequently, column I was replaced with column II. Column II separated dextran and N-glycan far better than the first column. In the process of the experiments a third column was established. Column III had the smallest porosity and diameter but at the same time it was also the longest column if the precolumn was taken into account. Column III showed the best results regarding peak intensities and peak efficiencies. Furthermore, solvent consumption was massively reduced through column III which used a flow rate of 100 µL min−1. Column III had the longest run time, 170 min vs. 105 min (column II) vs. 120 min (column I). However, column III did not need any runs with Milli-Q®-water between two samples as the required long run time ensured a proper washing time. Column III had three major advantages: It saves column lifetime, saves time, and had the best separation quality. On the other hand, LOD of column III was comparable to column II. While peak areas of column III decreased proportionally to concentration, peak areas of column II did not decrease consistently. This happened most likely to pipetting errors or handling mistakes. The detected LOD for both columns were down to single-digit pmol level (Fig 36). Samples used for the analysis of the LOD were not recovered from excessive 2AB label. At lower concentrations, the noise of 2AB label interfered with the N-glycan peaks. It is possible that purification of excessive fluorescent tag would enable detection of N-glycans at fmol level. This would be an important progress when it comes to the analysis of laser microdissected cells. Antigens were retrieved either through a trypsin or heat mediated process. In case of standard glycoproteins, it did not matter which antigen retrieval method was used. Nevertheless, differences in the chromatograms were observed as shown for human transferrin in Fig 35 and Fig 38. More (especially small) peaks were observed if the glycoproteins were treated with trypsin. There are two possible explanations for this outcome. •

The heat mediated approach purified N-glycans through its methanol-CHCI3-Milli-Q®water extraction. Samples treated with trypsin could contain peptide chains which interferes with the chromatogram.

•

The heat mediated approach cleaves SiaAc through its high temperature leaving only asialo-N-glycans and therefore, less peaks. 70

Discussion

To obtain this very valuable information, the heat mediated antigen retrieval has to be performed at different temperatures (e. g., 60°C, 80°C, and 100°C). This approach must also be performed with FFPE tissue sections. It hast to be proven, if temperatures under 100°C are sufficient for antigen retrieval of cross-linked proteins. The use of 10 kDa centrifugal filter (Tousi et al. 2013) after the N-glycan release could assist to purify samples even more. Ten kilodalton centrifugal filter would separate released Nglycans from PNGase F and large peptide chains already before RP-SPE with C18 tips.

5.2

N-Glycan Analysis of Whole FFPE Tissue Sections

N-glycans were only detected if the antigen retrieval was achieved through a heat mediated process. The question at this point is, why did the trypsin mediated approach fail to retrieve antigens from FFPE tissue sections but not from standard glycoproteins? Tryptic digestion could be enhanced by dispensing the trypsin solution in more aliquots. Multiple thawing of trypsin could decrease its activity. Another option is the use of another buffer or trypsin composition. Lastly, it can be assumed that trypsin was not inhibited by PMSF appropriately and therefore, PNGase F was proteolytic digested while it was used for cleaving N-glycans. After trypsin incubation overnight, samples could be incubated at 95 − 100°C for additional 10 min with mild agitation to destroy residual protease activity (Kolarich et al. 2015). If sialylated N-glycans could withstand 100°C for 1 h, heating for 10 min to inactivate trypsin should not be an issue. On the other hand, the heat mediated process led to proper antigen retrieval. Best results were obtained when N-glycans were released without prior dot-blotting (Fig 39). It hast to be stated, that positive results were achieved at the end of the experiments. Therefore, the analyses of whole FFPE tissue sections through various approaches were carried out only once. The experiments have to be repeated various times to evaluate all attempts precisely. Nonetheless, some significant observations were already made. Beside the advantages of the heat mediated process (especially achieving results), the pellet of higher protein amounts after the methanol-CHCI3-Milli-Q®-water precipitation are difficult to dissolve. Even if skipping the dot-blotting procedure led to higher peak intensities and efficiencies (so far), dot-blotting of (glyco)proteins onto PVDF membrane allows to release O-glycans after PNGase F digestion (Hinneburg et al. 2017). If one is interested in both, using the dot-blotting procedure might be an advantage. The protocol of deparaffinization in tubes could also be improved. The temperature could be increased to the melting point of paraffin (~ 56°C) and incubation on ice might be done at −20°C in order to remove waxy residues properly. Moreover, FFPE tissue sections can be deparaffinized in LoBind tubes and further processed in regular tubes as it has shown superior results compared to processing in LoBind tubes (Fig 39). This observation does make sense as the use of protein low-binding tubes lead to substantial losses of hydrophilic glycans (Jensen et al. 2012).

71

Discussion

5.3

N-Glycan Analysis from Laser Microdissected Cells

The analysis of laser microdissected cells caused more trouble as whole FFPE tissue sections. The noise of excessive 2AB label reagent interfered with the most peaks and therefore, made the analysis of laser capture microdissected sells impossible. All attempts with trypsin to retrieve antigens failed. The trouble shoot was already discussed in Section 5.2. It has to be discussed in which way the excessive 2AB label can be removed without losing major sample amounts. Off-line separations can be time consuming and cumbersome (Benet and Austin 2011), for which reason on-line may be superior. The first possibility is the use of an isocratic separation at the very beginning as it was mentioned in Section 4.4.2. If samples are considered to be analyzed with ESI-MS afterwards, it has to be assured that no free label reagent is collected with the peak fractions. For this reason, a trap column can be used as on-line SPE (Benet and Austin 2011) while the HPLC run starts with an isocratic separation (Fig 47). Fluorescent tags should be all trapped in the trap column.

Fig 47: Six-Port High-Pressure Valve with Trap Column for On-Line SPE. Abbreviations: SPE = solid phase extraction.

Nevertheless, off-line techniques like HILIC-SPE tips are still suitable methods to purify samples from excessive label reagent. This experiment failed most likely as N-glycans were still not recovered from DMSO. For this reason, one of the last experiments was to lyophilize an amount of 5 µL pure 2AB label reagent overnight in a dry freezer. Unlike all failed efforts trying to remove DMSO via centrifugal evaporation, DMSO was completely removed through lyophilization. Consequentially, 2AB labeled samples should not be dried in a centrifugal evaporator but in a dry freezer. HILIC-SPE purification can be tried once more and N-glycan should be adhered to the cotton wool this time. One more way to ensure proper elution is a higher amount of water to dissolve glycans from the cotton wool. It can be still assumed that N-glycans released from laser microdissected cells will not show high peak intensities. In this case, a more sensitive fluorescence detector shall be used. A 72

Discussion

more sensitive fluorescence detector already tested in this work. Unfortunately, the high amount of 2AB label caused a straight horizontal peak throughout the chromatogram (data not shown). Taken together, released N-glycans from at least ~1000 microdissected cells are a suitable amount for HPLC analysis. The experiments should continue with on-line purification (with or without trap column) and off-line purification after dry freezing 2AB labeled samples. As soon as excessive 2AB label reagents are removed, a more sensitive fluorescence detector may be considered to obtain higher peak intensities.

5.4

Analysis of Permethylated N-Glycans through Mass Spectrometry

Mass spectrometry of permethylated N-glycans were successful. Permethylation causes side reaction when small quantities of N-glycans are used (Zhou et al. 2017) which is the case for laser microdissected samples. Permethylation of N-glycans in very low pmol to fmol quantities may lead to oxidative degradation or peeling reactions which are associated with a high pH value resulting from dissolving NaOH powder (Kang et al. 2005). Those drawbacks can be overcome by changing the permethylation procedure, e. g., solid-phase permethylation (Zhou et al. 2017) or spin-column permethylation (Kang et al. 2008). N-glycans can still be under-, per-, or over-methylated which can be seen as serrate peaks in 14 m/z intervals. Permethylation is not the only solution for MS analysis of sialylated N-glycans. Another approach to enhance SiaAc stability involves selective modification of their carboxyl group. In contrast to permethylation, SiaAc linkages can be derivatized in a linkage specific manner (Bladergroen et al. 2015). α2,6-linkages undergo modifications such esterification, while α2,6linkages can form an intramolecular lactone (Holst et al. 2016), which leads to water loss. The reaction is shown in Fig 48.

Fig 48: Ethyl Esterification and Lactone Formation of Sialic Acids. A) 6’-sialyllactose (α2,6-linked sialic acid) shows ethyl esterification and B) 3’-sialyllactose (α2,3-linked sialic acid) shows lactone formation with neighboring galactose; taken from Reiding et al. (2014), page 5789.

73

Discussion

Other techniques, like methyl esterification or (methyl)amidation require harsh conditions, long reaction times, highly purified glycans, and lack in linkage specificity and/or complete derivatization (Reiding et al. 2014). A short protocol is given in the Appendix (Section 8.4.1).

5.5

Conclusion and Future Perspectives

LCM is a reliable method to isolate cells are areas of interest with minimal alteration of intracellular components (Cheng et al. 2013). Furthermore, it reduces any risks of contamination with other cells or tissue parts (Hinneburg et al. 2017). N-glycans on the cell surface are products of the same glycosylation machinery in the particular cell. Profiling those cells via LCM could give valuable information about how cells interact with other cells (Jensen et al. 2012). For a proper analysis of N-glycans thousands of cells have to be detached (Espina et al. 2006) which is a time-consuming process. LCM is not a fast or easy way to detach cells or areas of interest. Its use requires proper training and experience (Liu et al. 2014). Especially the cutting procedure needs patients. Huge areas cannot be cut easily (Emmert-Buck et al. 1996) as a vast amount of energy is needed to catapult cells. Once one has accomplished the task, LCM provides many opportunities and it is worth it. N-glycan analysis from microdissected cells in specific physiological or even pathophysiological states is a challenge as well as a promise for the future direction of glycomic research (Cummings and Pierce 2014). A single FFPE lung sample contains 168 sites for glycosylation (Ostasiewicz et al. 2010). Korekane et al. (2007) was the first group using LCM and successfully analyzing asialo Nglycans. However, the peak efficiency and sensitivity (Matsuda et al. 2008) of their approach was low. The second group published recently the use of LCM to analyze N- and O-glycans were Hinneburg et al. (2017). They needed the same number of cells per run to detect Nglycans. N-glycans were reduced and separated in a PGC column followed by ESI-MSn analysis. Glycans were detected in negative ion mode which has some disadvantages as described in Section 2.8. Without fluorescent labeling and permethylation or esterification their approach will not be as sensitive as the method introduced here as soon the excessive fluorescence tag is removed. Furthermore, the group did not select specific cells. Cells were microdissected in rectangles and unfortunately, no biological relevant information can be obtained from their results. Although the results in this work are not satisfying it is clear that a last purification step is needed to obtain significant results. Unfortunately, first peaks were achieved at the end of the thesis letting no time left to purify samples accordingly. On the other hand, sample recovery should be feasible. N-glycans from FFPE tissues are currently analyzed with other techniques than LCM. One popular approach is the use of MALDI-imaging mass spectrometry (IMS) (Heijs et al. 2016). MALDI-IMS technology has been advanced over the past decades and has enabled the development of high-throughput approaches to analyze multiple biomolecules in tissue sections simultaneously (Casadonte and Caprioli 2011, Turiak et al. 2014, Gustafsson et al. 2015). MALDI-IMS is a histology-based MS technique and allows the direct detection and visualization of biomolecules and their assignment to specific tissue areas in their native 74

Discussion

histological state. Tissue sections are mounted on compatible carrier, like indium tin oxide (ITO) coated glass slides. Mounted samples are coated with a matrix which absorbs and transfers laser energy to biomolecules (e. g., N-glycans) and therefore, facilitate desorption and ionization. (Ly et al. 2016). It is not as sensitive as immunohistochemistry (IHC) or fluorescence microscopy, but, on the other side, it does not require analyte with specific antibodies (Casadonte and Caprioli 2011). Powers et al. (2013) sprayed PNGase F directly on the tissue section mounted on histology glass slides and analyzed N-glycans with MALDI-IMS. Such techniques can be used for clinical approaches to distinct tumor from non-tumor tissues (Powers et al. 2014, Powers et al. 2015, Everest-Dass et al. 2016) or detect diseaseassociated N-glycans (Toghi Eshghi et al. 2014). All these approaches are suitable for clinical use and rapid diagnosis but they are not as precisely as LCM. LCM is rather a method for basic research. All in all, with the number of cells or tissue area needed, laser microdissection is a time-consuming procedure, especially if •

tissue sectioning, deparaffinization, and staining (1.5 d),

•

antigen retrieval (1 – 2 d)

•

N-glycan release and purification (1 d),

•

2AB labeling and purification (1 d),

•

HPLC analysis (1 d, depending on sample number),

•

permethylation or esterification followed by purification (1 d),

•

MS analysis (1 d, depending on sample number), and

•

structure determination (depending on knowledge)

are included. Therefore, it should be considered look for time saving approaches. One example is on-line purification of 2AB labeled N-glycans. Interestingly, Sandoval et al. (2007) reported an accelerated microwave-irradiation assisted PNGase F digestion that achieved complete deglycosylation in 30 min. As the human glycome or all glycan determinants are not known yet (Cummings and Pierce 2014), the field of glycobiology is still a long journey to go. The use of LCM might held to get more insight into the top-down level and coupled with bottom-up approaches would be helpful to define the relationship between glycans to their protein (and lipid) carriers.

75

Abstract

6

Abstract

Glycosylation of proteins is one of the most common co- and posttranslational modifications occurring in over 50% of all gene products. Almost all nuclear and DNA binding proteins, cytoplasmic enzymes, secreted proteins, membrane proteins and some mitochondrial proteins are glycosylated. N-glycans (linked to the amino acid asparagine) influence many properties of glycoproteins. For example, protein stability, solubility, antigenicity, half-life, clearance rate, in vivo activity, intracellular trafficking and facilitation of nascent polypeptide folding. Glycosylation appears cell specific and therefore, N-glycans may differ between cells. The analysis of N-glycans obtained from preserved formalin-fixed paraffin embedded (FFPE) tissues yields valuable information. On the other hand, FFPE tissue sections consist of heterogenous cells. For a better understanding of the role of N-glycans to its cells, homogenous subpopulation is needed. Laser capture microdissection (LCM) is typically used to overcome drawbacks of heterogenous cell samples by isolating specific cell/tissue regions. It enables the procurement of a microscopic and contaminationfree cellular section away from its heterogenous tissue milieu. The overall goal of this thesis was the development of a protocol for N-glycan analysis of laser microdissected cells from FFPE tissue sections. It was hypothesized that LCM of FFPE tissue sections is a suitable approach to analyze N-glycans of specific cells or areas of interest. The experiments started with the establishment of permethylation of (sialylated) N-glycans and their analysis using matrixassisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS). The study continued with normal phase (NP)-high performance liquid chromatography using a hydrophilic interaction liquid chromatography (HILIC) column. The aim was to establish the needed amount of microdissected cells, to test different antigen retrieval procedures, to optimize enzymatic N-glycan release, purification, as well as derivatization. Formalin induced crosslinkages were reversed through a heat mediated process. Consequently, Nglycans were enzymatically released, purified, fluorescence labeled and finally analyzed via HPLC. While 2-aminobenzamide (2AB) labeled N-glycans of whole FFPE tissue sections with a large area (150 mm2) were sufficiently detectable, lower amounts were interfered through baseline noise of 2AB. It was even more inconvenient when laser capture microdissected with very low areas (5.3 mm2 – 12.3 mm2) were analyzed. 2AB labeled N-glycan peaks were barely detectable. For this reason, HILIC-solid phase extraction (SPE) was implemented to overcome this issue and purify samples from excessive label reagent. Unfortunately, HILIC-SPE of excessive label reagent failed. It is assumed that all used centrifugal evaporator were not able to recover the samples from DMSO and therefore, allowing N-glycans to adhere at the cotton wool. Sample recovery may be achieved in a dry freezer. Alternatively, 2AB labeled N-glycans could be purified with an on-line SPE with or without using trapcolumn. LCM is a suitable technique for the analysis of N-glycans obtained from selected cells or tissue areas. As sample amounts are low and bioanalytical devices sensitive and expensive, no unnecessary detergents shall be used and samples have to be purified accordingly. With a few more modifications in the last purification steps it will soon be possible to obtain biological relevant information about the roles of N-glycans obtained from laser capture microdissected cells.

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7.1

Internet Links

https://www.carlroth.com/en/en/Life-Science/Histology-Microscopy/Clearing-Agents/XyleneAlternative-Roti%3Csup%3E%C2%AE%3C-sup%3E-Histol/Roti%3Csup%3E%C2%AE%3C-sup%3EHistol/p/000000010000330b00020023_en, last seen 12th Jan 2017 (Section 2.3.2). http://www.cellsignet.com/media/plates/96.jpg, last seen 23rd Nov 2016 (Fig 29 and Fig 31). http://www.fao.org/docrep/005/ac802e/ac802e0b.gif, last seen 23rd Nov 2016 (Fig 29 and Fig 31). http://obrazky.superia.cz/1280/otevrena_eppendorfova_zkumavka.png, last seen 23rd Nov 2016 (Fig 29). http://www.mackauraa.com/images/Vials-Caps-With-Septa.jpg, last seen 23rd Nov 2016 (Fig 31).

xii

Appendix

8

Appendix

8.1

Materials

8.1.1 Preparation of Dextran Hydrolysate

Tab 5: Materials Required for Dextran Hydrolysation.

Laboratory Balance

Manufacturer; Art.-No.

Lot-No.

Sartorius; BP221S

13510024

Ultrasonic Bath (+ Floating Tube Rack) Thermo Shaker

Kisker Biotech; TS-100C

Centrifugal Evaporator

Jouan; RC1022, or Christ; RVC 2-18 CDplus

Dextran from Leuconostoc spp.

Sigma Aldrich; 31392-10G

BCBK2600V

Roth; 6957.1

246238113

Forche Chromatographie; VT1100790

30082016

Trifluoroacetic Acid, ≥99.9% HPLC Glass Vial, 1.1 mL 8 mm Septum, Silicon, Shore A, 1.3 mm Safe-Lock Tubes 1.5 mL Milli-Q®-Water

Duratec Analysentechnik GmbH; 60103-0802 Eppendorf; 0030 120.086

D157823O

Millipore; F1HA81794D

2AB Solution (Section 3.5.4)

Abbreviations: 2AB = 2-aminobenzamid; HPLC = high performance liquid chromatography.

8.1.2 Preparation of Standard Glycoproteins

Tab 6: Materials Required for Preparation of Standard Glycoproteins

Laboratory Balance

Manufacturer; Art.-No.

Lot-No.

Sartorius; BP221S

13510024

Ultrasonic Bath (+ Floating Tube Rack) pH Meter, inoLab Ammonium Bicarbonate

WTW Fluka Analytical; 09830-1KG

BCBB4824

Ammonia Solution 25% Suprapur® for pH adjustment

Merck; 1054280250

Asialofetuin from Fetal Calf Serum

Sigma Aldrich, A-4781

93H9506

Sigma Aldrich, F3385-100MG

078K43

Fetuin from Fetal Bovine Serum Ribonuclease B from Bovine Kidney Apo-Transferrin Human SafeSeal Micro Tube 0.5 mL Milli-Q®-Water

Sigma Aldrich; R-7883 Sigma Aldrich; T2036-100MG

SLBG1252V

Sarstedt; 72.704.400

6081511

Millipore; F1HA81794D

xiii

Appendix

8.1.3 Cutting Procedure

Tab 7: Material List for Cutting Procedure via Microtome.

Microtome, RM2165

Manufacturer; Art.-No.

Lot-No.

Leica; 050331621

0148/08.1998

Heating Oven

Memmert

Heating Bath

Medax Type 25900; 35326

198-242

Heating Plate

Leica HI1220; 042321474

2337/02/2000

SuperFrost®

R. Langenbrinck Plus Objektträger; 03-0060

022515

MembraneSlides 1.0 PEN

Zeiss; 415190-9041-000

000741-12

Safe-Lock Tubes 1.5 mL

Eppendorf; 0030 120.086

D157823O

Protein LoBind Tube 1.5 mL

Eppendorf; 0030 108.116

B146225H

Histology Glass Slides

Razor Blades Distilled Water

Abbreviations: PEN = polyethylene naphthalate.

8.1.4 Deparaffinization and Staining on PEN and Histology Glass Slides

Tab 8: Material List for Deparaffinization and Staining on Histology Glass Slides. Manufacturer; Art.-No.

Lot-No.

Eppendorf; 0030 120.086

D157823O

Roti®-Histol

Roth; 6640.4

312189427

2-Propanol, 99.5% for Synthesis

Roth; 9866.5

366249199

Hemalum Solution Acid According to Mayer

Roth; T865.2

525238820

Eosin Y Solution 0.5% in water

Roth; X883.2

030152858

Roti®-Mount Aqua, Density 1.05 − 1.18

Roth; 2848.1

186235967

Cover Slips, 24 x 40 mm

Roth; 1870.2

1870

Coplin Jars Coplin Racks Safe-Lock Tubes 1.5 mL

Needle/Pin Distilled Water

xiv

Appendix

8.1.5 Deparaffinization in Tubes

Tab 9: Material List for Deparaffinization in Tubes. Manufacturer; Art.-No. Thermo Shaker Centrifuge, UEC Micro 14/B

Lot-No.

Kisker Biotech; TS-100C UniEquip; C0160B-UE-230

812512

Vortex Mixer, Vortex V-1 Plus

Kisker Biotech; 144200B

Protein LoBind Tube 1.5 mL

Eppendorf; 0030 108.116

B146225H

Safe-Lock Tubes 1.5 mL

Eppendorf; 0030 120.086

D157823O

Paraplast® Plus

Leica; 39602004

Roti®-Histol

Roth; 6640.4

312189427

2-Propanol, 99.5% for Synthesis

Roth; 9866.5

366249199

Styrofoam Box Filled with Crushed Ice Distilled Water

8.1.6 Laser Capture Microdissection

Tab 10: Material List for Laser Capture Microdissection. Manufacturer; Art.-No. PALM CombiSystem (Containing PALM MicroBeam and PALM MicroTweezers, Power Supply 231, AxioCam ICc 1, and HXP 120 C, CapMover, HAL 100)

Zeiss; 1021.579134 4/5

TubeCollector 2x200 CM II

Zeiss; 415101-2000-410

SlideHolder 3x1.0 for MembraneSlides 1.0 PEN

Zeiss; 415101-2000-810

PALM Robo Software 4.5 Pro AdhesiveCap 500 opaque, 50 pieces Personal Computer with two Displays; one as Touchscreen (with Smart Stylus) Parafilm

Lot-No.

Zeiss Zeiss; 415190-9201-000

000630-16 000831-16

Touchscreen and Pen: Wacom Roth; H666.1

Abbreviations: PALM = photoactivated localization microscopy; PEN = polyethylene naphthalate.

xv

Appendix

8.1.7 Trypsin Mediated Antigen Retrieval Tab 11: Material List for Trypsin Mediated Antigen Retrieval. Manufacturer; Art.-No. Centrifugal Evaporator, RC1022

Lot-No.

Jouan

Ultrasonic Bath (+ Floating Tube Rack) Thermo Shaker

Kisker Biotech; TS-100C

Palm Micro Centrifuge, D1008

Dragonlab

2 L beaker Bio Magnetic Stirrer (+Stirrer Magnet)

Hartenstein; MMS-3000

Slide-A-Lyzer® MINI Dialysis Units, 50 Units Safe-Lock Tubes 1.5 mL SafeSeal Micro Tube 0.5 mL

Thermo Fisher Scientific, 69590

NJ177389

Eppendorf; 0030 120.086

D157823O

Sarstedt; 72.704.400

6081511

Roth; 9866.5

366249199

Roth; 6331.3

501178788

Thermo Scientific; 36978

NK1624004

2-Propanol, 99.5% for Synthesis Hydrochloric Acid,

Rotipuran®

730702079

≥25%

Trypsin Phenylmethylsulfonylfluoride ≥25%

8.1.8 Heat Mediated Antigen Retrieval Tab 12: Material List for Heat Mediated Antigen Retrieval. Manufacturer; Art.-No. Sonicator, Labsonic® M

Lot-No.

Sartorius

Ultrasonic Bath (+ Floating Tube Rack) Stereo Microscope

Olympus; SZ-STP

Cold Light Source

Zeiss; KL 1500 LCD

Vortex Mixer, Vortex V-1 Plus

Kisker Biotech; 144200B

Thermo Shaker

Kisker Biotech; TS-100C

Centrifuge, UEC Micro 14/B (for 2,000 g)

UniEquip; C0160B-UE-230

Centrifuge, Biofuge 1.0R (for ≥ 14,000 g)

Thermo Fisher Scientific

pH Meter, inoLab

812512

WTW

SafeSeal Micro Tubes 2 mL

Sarstedt; 72.695.500

0771/6243013

Eppendorf; 0030 120.086

D157823O

Sodium Dodecyl Sulfate Pellets ≥99%,

Roth; CN30.2

354212849

Tris, Pufferan® ≥99,9%, Ultra Quality

Roth; 5429.3

272186614

1,4-Dithiothreitol

Roth; 6908.1

01568801

Hydrochloric Acid, Rotipuran® ≥25%,

Roth; 6331.3

501178788

Chloroform, Rotisolv® HPLC

Roth; 7331.2

1073571

Roth; 4627.4

16786942

Safe-Lock Tubes 1.5 mL

Methanol,

Rotipuran®

PlusOne Urea

≥99,9%

GE Healthcare Life Sciences; 11K280022

Milli-Q®-Water

xvi

Appendix

8.1.9 N-Glycan Release from Dot-Blotted Glycoproteins onto PVDF Membranes

Tab 13: Material List for Dot-Blotting of (Glyco)Proteins. Manufacturer; Art.-No. Heating oven

Lot-No.

Memmert

Well Plate Centrifuge

mLw; T 62.1

Ultrasonic Bath (+ Floating Tube Rack) Gyratory Shaker, Mini Rocker

BioSan; Bio MR-1

Glass Petri Dish Kimtech wipes, 20.5 x 20 cm PVDF membrane, 0.45 µm

Kimtech Sciences; 7558 Merck Millipore; IPVH00010

K4JA4939D

Sarstedt; 82.1581

5023611

Cleaned Pair of Scissors Plastic Tweezer 96-well plate Falcon Tube, 50 mL SafeSeal Micro Tube 0.5 mL Safe-Lock Tubes 1.5 mL

Greiner Bio-One; 227261 Sarstedt; 72.704.400

6081511

Eppendorf; 0030 120.086

D157823O

Parafilm

Roth; H666.1

Coomassie® Brilliant Blue R 250

Serva

05062

Roth; 4627.4

16786942

Merck Millipore; 1000660250

812512

Sigma Aldrich; PVP40-50G

WXBC1374V

New England Biolabs; P0705L Roche; 11365193001

10663420

Methanol, Rotipuran® ≥99,9% Suprapur Acetic Acid (glacial) 100% Polyvinylpyrrolidone, PVP40 PNGase F, 500,000 U mL−1 PNGase F, 250 U mL−1 Milli-Q®-Water

8.1.10 N-Glycan Release in Solution

Tab 14: Material List for N-Glycan Release in Solution. Manufacturer; Art.-No. Thermo Shaker Palm Micro Centrifuge, D1008

Lot-No.

Kisker Biotech; TS-100C Dragonlab

Ultrasonic Bath (+ Floating Tube Rack) Safe-Lock Tubes 1.5 mL PNGase F, 500,000 units mL−1 PNGase F, 250 units mL−1

Eppendorf; 0030 120.086

D157823O

New England Biolabs; P0705L Roche; 11365193001

10663420

Milli-Q®-water

xvii

Appendix

8.1.11 N-Glycan Purification

Tab 15: Material List for N-Glycan Purification. Manufacturer; Art.-No. Palm Micro Centrifuge, D1008

Centrifugal Evaporator Pierce® C18 Tips, 100 µL

Dragonlab Jouan; RC1022, Christ; RVC 2-18 CDplus, or UniEquip; Univapo 150 H Thermo Scientific; 87784

RF234355

Sarstedt; 82.1581

5023611

Eppendorf; 0030 120.086

D157823O

96-Well Plate Safe-Lock Tubes 1.5 mL Methanol,

Rotisolv®,

Lot-No.

HPLC Gradient

Roth; KK39.2

Trifluoroacetic Acid, ≥99.9%

Roth; 6957.1

246238113

Acetonitrile, Rotisolv®, HPLC Gradient

Roth; HN44.2

A16A031621015

Milli-Q®-Water

8.1.12 Derivatization with 2-Aminobenzamide

Tab 16: Material List for Labeling with 2-Aminobenzamide. Manufacturer; Art.-No.

Centrifugal Evaporator, RC1022

Palm Micro Centrifuge, D1008

Lot-No.

Jouan; RC1022, Christ; RVC 2-18 CDplus, or UniEquip; Univapo 150 H Dragonlab

Ultrasonic Bath (+ Floating Tube Rack) Thermo Shaker

Kisker Biotech; TS-100C

Safe-Lock Tubes 1.5 mL

Eppendorf; 0030 120.086

D157823O

Dimethyl Sulfoxide

Sigma Aldrich; D8418-50ML

84596JMV

Suprapur Acetic Acid (Glacial) 100%

Merck Millipore; 1000660250

607002006

Merck; 820065

340102762

Sigma Aldrich; 15,615-9

S43382-307

2-Aminobenzamide Sodium Cyanoborohydride, 95% Milli-Q®-water

xviii

Appendix

8.1.13 Purification of N-Glycans from Excess 2-Aminobenzamide

Tab 17: Material List for Purification of Excess 2-Aminobenzamide. Manufacturer; Art.-No. Centrifugal Evaporator

Lot-No.

Jouan; RC1022, or Christ; RVC 2-18 CDplus

Ultrasonic Bath (+ Floating Tube Rack) 100% Cotton Wool 96-Well Plate SafeSeal Micro Tube 0.5 mL HPLC Glass Vial, 1.1 mL 8 mm Septum, Silicon, Shore A, 1.3 mm

Sarstedt; 82.1581

5023611

Sarstedt; 72.704.400

6081511

Forche Chromatographie; VT1100790

30082016

Duratec Analysentechnik GmbH; 60103-0802

Trifluoroacetic Acid, ≥99.9%

Roth; 6957.1

246238113

Acetonitrile, Rotisolv®, HPLC Gradient

Roth; HN44.2

A16A031621015

Milli-Q®-Water

xix

Appendix

8.1.14 High Performance Liquid Chromatography

Tab 18: Material List for HPLC Analysis. Manufacturer; Art.-No. Smartline Pump 1000

Knauer; V 7603 10/2007

Smartline Manager 5000 with Integrated Degasser

Knauer; V 7602 10/2007

Manual Injector A/D converter

Lot-No.

Rheodyne; Model 8125 (20 µL) Rheodyne; Model 7125 (50 µL) Knauer

Hamilton Syringe (25 µL and 50 µL) Autosampler Type 830

Midas; 0060.163-15

Fluorescence Detector

Merck Hitachi; F-1050

Column Oven Personal Computer with Installed Software EuroChrom for Windows 3.05 HILIC-Column I, TSKgel® Amide-80 Å, 4.6 mm x 25.0 cm

Knauer Tosoh Bioscience; 8AM286PHR0144

13071

HILIC-Column II; SeQuant® ZIC®-HILIC, PEEK Coated HPLC Column, 3.5 µm, 200 Å; 150 x 4.6 mm

Merck; 1.50449.0001

TA1877176

HILIC-Column III; SeQuant® ZIC®-HILIC, PEEK Coated HPLC Column, 3.5 µm, 100 Å; 250 x 2.1 mm

Merck; 1.50443.0001

TA1941375

Pre-Column; SeQuant® ZIC®-HILIC, PEEK Coated HPLC Column, 3.5 µm, 100 Å; 20 x 2.1 mm

Merck; 1.50439.0001

HPLC Glass Vial, 1.1 mL

8 mm Septum, Silicon, Shore A, 1.3 mm

Forche Chromatographie; VT1100790

30082016

Duratec Analysentechnik GmbH; 60103-0802

Centrifuge, Megafuge 15 R (for HPLC glass vials)

Thermo Fisher Scientific

Palm Micro Centrifuge, D1008 (for 1.5 mL tubes)

Dragonlab

Ultrasonic Bath (+ Floating Tube Rack) Schott/Duran Flask, 1 L Measuring Cylinder Falcon Tube, 50 mL

Greiner Bio-One; 227261

Acetonitrile, Rotisolv®, HPLC Gradient

Roth; HN44.2

2-Propanol, Rotisolv®, HPLC

Roth; 7343.1

Ammonium acetate, ≥ 97%

Roth; 7869.3

Ammonium formate, for HPLC ≥ 99.0% Hydrochloric Acid, Rotipuran® ≥25% pH adjustment of ammonium formate (Section 8.5.12)

A16A031621015

Fluka Analytical; 17843-50G

BCBL2129V

Roth; 6331.3

501178788

Milli-Q®-Water

Abbreviations: A/D = analog/digital; HILIC = hydrophilic interaction liquid chromatography; HPLC = high performance liquid chromatography; PEEK = polyether ether ketone; ZIC = zwitterionic.

xx

Appendix

8.1.15 Permethylation

Tab 19: Material List for Permethylation of N-Glycans. Manufacturer; Art.-No.

Lot-No.

Jouan; RC1022, or Christ; RVC 2-18 CDplus

Centrifugal Evaporator Ultrasonic Bath (+ Floating Tube Rack) Exsiccator Glass Vials (3 − 5 mL) 16 mm Septum, Silicon, Shore A, 1.9 mm

Duratec Analysentechnik GmbH; 60368-1602

Thermo Shaker Glass Vial Rack for vortexing multiple samples

Kisker Biotech; TS-100C

Vortex Mixer, Vortex V-1 Plus

Kisker Biotech; 144200B

Centrifuge, Megafuge 15 R

Thermo Fisher Scientific Forche Chromatographie; VT1100790

HPLC Glass Vial, 1.1 mL

8 mm Septum, Silicon, Shore A, 1.3 mm Dimethyl Sulfoxide, Hybri-MaxTM, ≥ 99.7% Sodium Hydroxide, Pellets Iodomethane,

ReagentPlus®,

29218

30082016

Duratec Analysentechnik GmbH; 60103-0802 Sigma Aldrich; D2650-5x5ML

RNBD8010

Roth; 6771.1 99%

Sigma Aldrich; 18507-100ML

Styrofoam Box Filled with Crushed Ice Milli-Q®-Water Acetonitrile, Rotisolv®, HPLC Gradient only for Purification (Section 8.4.1)

Roth; HN44.2

Methanol, Rotisolv®, HPLC Gradient only for Purification (Section 8.4.1)

Roth; KK39.2

Pierce® C18 Tips, 100 µL only for Purification (Section 8.4.1)

Thermo Scientific; 87784

A16A031621015

RF234355

xxi

Appendix

8.1.16 Mass Spectrometry

Tab 20: Material List for MALDI-TOF-MS. Manufacturer; Art.-No. Ultraflex TOF/TOF MALDI

Bruker

Personal Computer with Installed Software flexControl (3.4) and flexAnalysis (3.3)

Bruker

AnchorChip Target with 384 Spots

Bruker

Lot-No.

Ultrasonic Bath (Floating Tube Rack for Samples) Palm Micro Centrifuge, D1008 Safe-Lock Tubes 1.5 mL Acetonitrile, Rotisolv®, HPLC Gradient

Dragonlab Eppendorf; 0030 120.086

D157823O

Roth; HN44.2

A16A031621015

2,5-Dihydroxybenzoic Acid Milli-Q®-Water Kimtech wipes, 20.5 x 20 cm only for Cleaning (Section 8.4.2)

Kimtech Sciences; 7558

Methanol, Rotipuran® ≥99,9% only for Cleaning (Section 8.4.2)

Roth; 4627.4

Ethanol, ≥99,5% only for Cleaning (Section 8.4.2)

16786942

Roth; 5054.3

800 mL Beaker only for Cleaning (Section 8.4.2)

8.2

Additional Figures

Fig 49: Nomenclature for Carbohydrate Fragments. A, B and C fragments maintain the charge on the non-reducing end. X, Y and Z retain the charge on the reducing end. B, C, Y and Z correspond to glycosidic linkages while A and X fragments correspond to cross-ring cleavages. The subscript numbers denote the cleavage position. Source: Morelle and Michalski (2007), page 1589.

xxii

Appendix

Fig 50: Laser Capture Microdissected Sperms from Ductus Epididymis (Roe Deer Deceased in August 2001). A shows selected sperms from Ductus epididymis in 20-fold magnification while B displays the detached lumen in 20-fold magnification. C shows microdissected cells adhered to the AdhesiveCap in 5-fold magnification.

xxiii

Appendix

8.3

Additional Tables

Tab 21: Consensus Motifs and Glycan Linkages. Type N-glycosylation

Linkage GlcNAc-β-Asn

Consensus Sequence Asn-X-Ser/Thr (standard sequence) Asn-X-Cys, Asn-Gly, Asn-X-Val (non-standard sequence)

O-glycosylation

C-mannosylation

Examples nascent polypeptides

GalNAc-αSer/Thr

isoform specific

mucins

GlcNAcβ‑Ser/Thr

not set

cytoplasmic/ nuclear proteins

Xyl‑β‑Ser

Asp/Glu- Asp/Glu- Asp/Glu- Asp/Glu-Gly-SerAsp/Glu-Asp/Glu/Gly-Asp/Glu

Heparin

Fuc‑α‑Ser/Thr

Cys-X-X-Ser/Thr-Cys-X-X-Gly

thrombospondin

Cys-X X-X-X-Ser/Thr-Cys

Notch

Glc‑β‑Ser

Cys-X-Ser-X-Ala/Pro-Cys

Notch

Man‑α‑Ser/Thr

Ile-X-Pro-Thr-Z-Thr-X-Pro-X-X-X-X-Pro-Thr-XT/X-X

α‑dystroglycan

Gal‑β‑Hyl

X-Hyl-Gly

collagen

Glc‑α‑Tyr

Tyr194

glycogenin

Man-α-Trp

Trp-X-X-Trp

thrombospondin

Abbreviations: Ala = alanine; Asn = asparagine; Asp = aspartic acid; Cys = cysteine; Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc = glucose; GlcNAc = N-acetylglucosamine; Glu = glutamic acid; Gly = glycine; Hyl = hydroxylysine; Ile = isoleucine; Man = mannose; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Tyr = tyrosine; Val = valine; X = any amino acid except proline; Xyl = xylose; Z = any amino acid. Source: Moremen et al. (2012), page 449.

xxiv

Appendix Tab 22: Mammalian Glycan Linkages produced by Glycosyltransferases. Donors

Acceptors

Fuc

Gal

GlcNAc

GDP-Fuc

α1,2

UDP-Gal

α1,3 α1,4 β1,3

β1,3

UDP-GlcNAc

α1,3 β1,3 β1,4

α1,3 α1,6

Glc

GalNAc

β1,4

β1,4

α1,2

UDP-GalNAc

β1,3

β1,3 β1,6

β1,6

α1,6 β1,4

β1,3 β1,4

β1,3

β1,3 β1,4

UDP-Xyl

Xyl

β1,4

β1,4

α1,2 α1,3

α1,3

α1,4 β1,4

GDP-Man

α2,3 α2,6

SiaAc

β1,3 β1,4

β1,3

CMP-SiaAc

Man

α1,3 α1,4 α1,6

UDP-Glc

UDP-GlcA

GlcA

α2,6

α1,4 β1,4

β1,2

α1,2 α1,3 α1,6 α2,8

α1,3

α1,3

Abbreviations: CMP = cytidine 5’-monophosphate; Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GDP = guanosine 5’-diphosphate; Glc = glucose; GlcA = glucuronic acid; GlcNAc = N-acetylglucosamine; Man = mannose; SiaAc = sialic acid; UDP = uridine-5’-diphosphate; Xyl = xylose. Source: Ohtsubo and Marth (2006), page 855.

xxv

Appendix Tab 23: Expected N-Glycans from Bovine Fetuin B.

GlcNAc

Man

Gal

Neu5Ac

Neu5Gc

Fuc

N-glycan structures were created by GlycoWorkbench (Ceroni et al. 2008, Damerell et al. 2012). Note: Further isoforms through α2,3- and α2,6-linkages of Neu5Ac and Neu5Gc are not shown. Abbreviations: Fuc = fucose; Gal = galactose; GlcNAc = N-acetylglucosamine; Man = mannose; Neu5Ac = N-acetylneuramic acid; Neu5Gc = N-glycolylneuramic acid. Source: Aich et al. (2013), page 3.

xxvi

Appendix Tab 24: Expected N-Glycans from Ribonuclease B.

GlcNAc

Man

N-glycan structures were created by GlycoWorkbench (Ceroni et al. 2008, Damerell et al. 2012). Abbreviations: GlcNAc = N-acetylglucosamine; Man = mannose Source: Fu et al. (1994) and Prien et al. (2009).

xxvii

Appendix Tab 25: Expected N-Glycans from Human Transferrin.

GlcNAc

Man

Gal

Neu5Ac

Fuc

N-glycan structures were created by GlycoWorkbench (Ceroni et al. 2008, Damerell et al. 2012). Note: An increased number of tri- and tetra-antennary N-glycans as well as higher fucosylation was reported in patients with carbohydrate deficient transferrin due to alcohol abuse. Tetra-antennary Nglycans are not shown in this table. Abbreviations: Fuc = fucose; Gal = galactose; GlcNAc = N-acetylglucosamine; Man = mannose; Neu5Ac = N-acetylneuramic acid. Source: Landberg et al. (2012), Nagae et al. (2014), and Quaranta et al. (2016).

xxviii

Appendix Tab 26: Examples of Monosaccharide Masses. Classification

Super Classes

Unsubstituted sugars

Amino sugars

Deoxy sugars

Methylated sugars

Acidic sugars

Native Masses

Substitution

Accurate

Average

Permethylation

Peracetylation

Hexose

162.053

162.141

3

3

Pentose

132.042

132.115

2

2

Heptose

192.063

192.167

4

4

2-Hexosamine

161.069

161.157

4

3

2-N-Acetylhexosamine

203.079

203.193

3

2

6-Deoxyhexose

146.058

146.142

2

2

5-Deoxypentose

116.048

116.116

4

1

4,6-Dideoxyhexose

130.063

130.142

4

1

3-O-Methylhexose

176.068

176.168

2

2

4-O-Methylhexose

176.068

176.168

2

2

Hexuronic acid

176.032

176.125

3

3

N-Acetylneuraminic acid

291.095

291.256

5

4

N-glycolylneuraminic acid

307.090

307.255

6

5

KDN

250.069

250.204

5

5

KDO

220.058

222.178

4

4

Muramic acid

275.100

275.256

3

2

Incremented masses of monosaccharides minus the mass of water. Abbreviations: KDN = 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid; KDO: 3-deoxy-D-manno-oct-2ulopyranosonic acid. Sources: Maass et al. (2007), page 4436.

Tab 27: Common Encountered B-Type Ions. B-Type Ion

Mass

B-Type Ion

Mass

HexNAc1 + Na+

282

HexNAc2Fuc1 + Na+

701

NeuAc1 + Na+

398

HexNAc1Hex1HexNAc1 + Na+

731

HexNAc1Hex1 + Na+

486

HexNAc1Hex1Fuc2 + Na+

834

HexNAc2 + Na+

527

HexNAc1Hex1NeuAc1 + Na+

847

HexNAc1Hex1Fuc1 + Na+

660

HexNAc2Hex2 + Na+

HexNAc1Hex2 +

Na+

690

HexNAc1Hex1Fuc1NeuAc1 +

935 Na+

1.021

Masses correspond to non-reduced structures in sodiated permethylated glycans. Abbreviations: Fuc = fucose; Hex = hexose; HexNAc = N-acetylhexoseamine; NeuAc = N-acetyl/glycolyl-neuramic acid. Source: Morelle and Michalski (2007), page 1590.

xxix

Appendix Tab 28: Residue Masses of Common Terminal Groups. Terminal Groups

Native Masses

Permethylated Masses

Accurate

Average

Accurate

Average

Non-reducing end

1.008

1.008

15.024

15.035

Reducing end

17.003

17.007

31.018

31.034

Reduced reducing end

19.018

19.023

47.050

47.077

Sum of mass with the sodium for N-glycans

41.000

41.014

69.032

69.068

Sum of mass with the sodium for O-glycans

43.016

43.030

85.063

85.111

Sources: Morelle and Michalski (2007), page 1588.

Tab 29: Commonly Used Exoglycosidases and Incubate Conditions. Enzyme, Source

Specificity

Buffer, pH

Concentration, Incubation Time

α2,3, α2,6, α2,8 SiaAc

50 mM sodium acetate, pH 5

1 µU µL−1 3− 16 h

α2,3 SiaAc

50 mM sodium phosphate, pH 6.0

1 µU µL−1 16 h

β-Galactosidase [EC 3.5.1.52], Bovine testes

β1,3 and β1,4 Gal

50 mM ammonium formate, pH 4.6

1 mU µL−1 3− 16 h

β-Galactosidase [EC 3.2.1.23], Streptomyces pneumoniae

β1,3 Gal

100 mM sodium acetate, pH 6.0

80 µU µL−1 16 h

β1,3 and β1,4 GlcNAc or GalNAc

100 mM sodium citratephosphate, pH 6.0

10 µU µL−1 16 h

β-N-Acetylglucosaminindase [EC 3.2.1.30], Streptomyces pneumoniae

β1,3 and β1,4 GlcNAc

100 mM sodium citratephosphate, pH 6.0

120 µU µL−1 16 h

α-Fucosidase [EC 3.2.1.111], Almond meal

α1,3 Fuc

50 mM sodium acetate, pH 6.0

1 mU µL−1 16 h

α-Fucosidase [EC 3.2.1.51], Bovine kidney

α1,4 and α1,6 Fuc

100 mM sodium citrate, pH 6.0

1 mU µL−1 16 h

α-Mannosidase [EC 3.2.1.23], Jack bean

α1,2 and α1,4 Man

100 mM sodium acetate, 2 mM Zn, pH 5.0

67 mU µL−1 2 x 16 h

α-Mannosidase [EC 3.2.1.24], Bovine kidney

α1,2, α1,3 and α1,6 Man

50 mM ammonium acetate, pH 4.5

67 mU µL−1 2 x 16 h

Endo-β-galactosidase [EC 3.2.1.102], Bacteroides fragilis

β1,3 and 1,4 Gal in poly Nacetyllactosamine

50 mM ammonium acetate, pH 5.8

100 µU µL−1 3h

α-Sialidase [EC 3.2.1.18], Arthrobacter ureafaciens Sialidase [EC 3.2.1.16], Newcastle disease virus

β-N-Acetylhexosaminidase [EC 3.2.1.23], Recombinant

Far more exoglycosidase are available. For appropriate sequencing knowledge of acceptor structure is indispensable. Abbreviations: Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GlcNAc = Nacetylglucosamine; Man = mannose; SiaAc = sialic acid. Source: Morelle and Michalski (2007), page 1590 and Merry and Astrautsova (2009), page 209.

xxx

Appendix

8.4

Additional Protocols

8.4.1 Purification of Permethylated N-Glycans The purification procedure was adapted from Morelle and Michalski (2007). Similar to the other N-glycan purification steps (Sections 3.5.3 and 3.5.5) this protocol can be carried out with a regular pipette or by a multichannel pipette for treatment of multiple samples at once. The following solutions were prepared for purification of permethylated N-glycans. •

Wetting and Conditioning solution: 500 µL methanol.

•

Washing solutions: 1.5 mL Milli-Q®-water and 300 µL 10% ACN.

•

Elution solution: 300 µL 80% ACN.

Dried samples were dissolved in 100 µL methanol. Methanol was resuspended along the inside of the HPLC glass vial to ensure that all permethylated N-glycans were uptaken. C18 tips were conditioned five times with 100 µL methanol. Next, samples were slowly aspirated and dispensed 30 times using a C18 tip in order to allow N-glycan adsorption. C18 tips were washed 15 times with 100 µL Milli-Q®-water and three times with 100 µL 10% ACN. Permethylated Nglycans were eluted with 300 µL 80% ACN and transferred into new HPLC glass vials. Samples were dried in a centrifugal evaporator and stored at −20°C until MS analysis (Section 3.8).

8.4.2 Cleaning of the Stainless Steel AnchorChip Target This protocol is adapted from Signor and Boeri Erba (2013). The AnchorChip target was rinsed in methanol and wiped gently by using a Kimtech wipe. This procedure was repeated once with Milli-Q®-water. The AnchorChip was inserted into a 800 mL beaker and completely covered with 50% ethanol. Target was sonicated for 10 min in an ultrasonic bath. The target was rinsed again with methanol and tilted in a way that the complete liquid is collected on a Kimtech Wipe. Finally, the target was air-dried at RT.

8.4.3 Esterification of Linkage Specific Esterification of Sialic Acids This protocol is mainly adapted from Reiding et al. (2014) and Bladergroen et al. (2015). Esterification may be performed after a first HPLC run when all glycan peaks are identified (Section 3.6). After derivatization, sialylated glycans may show shifted glycan peaks in the chromatogram. First, all glycan peaks of each run should be collected, either manually or via sample collector. Collected samples are lyophilized prior derivatization. 20 µL of ethanol containing 0.25 M 1ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and 0.25 M 1-hydroxybenzotriazole hydrate (HOBt) are added to dried and purified 2AB labeled glycans (after completing Section or ). Samples are incubated for 1 h at 60°C. After finishing the heat incubation, 20 µL of HPLC gradient solution containing 75% ACN and 25% NH4CH3CO2 are added to each sample and incubated for 15 min at −20°C. Samples were put into the sonicator bath for 3 min and can be analyzed by HPLC following by MS. The linkages can certainly be discriminated by mass changes. xxxi

Appendix

8.5

Calculations

8.5.1 Trifluoroacetic Acid (0.1 M) TFA: M = 114.02 g mol−1; V = 0.001 L; c = 0.1 M; ρ = 1.48 g L–1 •

(114.02 g mol−1 x 0.001 L x 0.1 mol L−1) / 1.48 g cm−3 = 7.8 µL

•

7.8 µL TFA was added to 992.2 µL Milli-Q®-water.

TFA was stored at 4°C until usage.

8.5.2 Ammoniums Bicarbonate (25 mM, pH 8.0) NH4HCO3: M = 79.06 g mol−1; V = 0.04 L; c = 0.025 M •

m = 0.025 mol L−1 x 0.04 L x 79.06 g mol−1 = 79.06 mg

•

79.06 mg was transferred into a measuring cylinder (50 mL).

•

Milli-Q®-water was added to reach a final volume of 40 mL.

•

25 mM NH4HCO3 was transferred into a 50 mL Falcon tube and vigorously vortexed until the salt was completely dissolved.

•

A couple of ammonia drops were added to achieve a pH of 8.0.

NH4HCO3 was stored at RT but not longer than 2 weeks as it changes its pH value over time (Hinneburg et al. 2017).

8.5.3 Trypsin (1 mg mL−1) in 0.001 N Hydrochloric Acid HCl: M = 36.46 mg L−1; ρ = 1.18 g L–1; 25% •

0.001 N HCl = 0.03646 g L−1 / 1.18 g L−1 = 30.9 µL L−1 (100%) = 123.6 µL L−1 (25%)

•

1.3 µL HCl (25%) was added to −9.999 mL Milli-Q®-water.

•

mg Trypsin was transferred into a 1.5 mL tube.

•

mL of 0.001 N HCl was added to Trypsin.

•

Tube was sonicated in ultrasonic bath for 3 min using a floating tube rack.

Trypsin was stored at −20°C until usage.

8.5.4 Phenylmethylsulfonylfluoride (100 mM) PMSF: M = 174.2 g mol–1; V = 0.01 L, c = 0.1 M •

m = 0.1 mol L–1 x 0.01 L x 174.2 g mol–1 → 174.2 mg

•

174.2 mg PMSF was transferred into a 15 mL Falcon tube.

•

10 mL of isopropanol was added.

•

Falcon tube was sonicated in ultrasonic bath for 3 min using a floating tube rack.

100 µL aliquots of PMSF solution in 0.5 mL tubes were stored at −20°C until usage. xxxii

Appendix

8.5.5 Tris-Hydrochloride (0.1 M, pH 8) Containing 4% Sodium Dodecyl Sulfate Tris-HCl: M = 121.14 g mol–1; V = 0.1 L, c = 0.1 M •

m = 0.1 mol L–1 x 0.1 L x 121.14 g mol–1 → 1.2114 g

•

1.2114 g Tris-HCl was transferred into a volumetric flask.

•

80 mL Milli-Q®-water was added and shaken gently until Tris was solved completely.

•

~ 9 mL HCl (25%) was added until pH was adjusted to 8.

•

~ 11 mL Milli-Q®-water was added to reach a final volume of 100 mL.

•

Solution was then transferred in a Schott/Duran flask and 4 g of SDS was added.

•

Flask was put on a hot plate stirrer until SDS was solved through stirring.

Tris-HCl containing 4% SDS was stored at 4°C at the most of two weeks (Hinneburg et al. 2017). SDS precipitates over time at 4°C. Before usage SDS was solved on a stirrer magnet.

8.5.6 Dithiothreitol (0.1 M) DTT: M = 154.2 g mol–1; V = 2.0 mL; c = 0.1 M •

m = 0.1 mol L–1 x 0.002 L x 154.2 g mol–1 → 30.84 mg

•

30.84 mg DTT was added into a 2.0 mL tube.

•

1.5 mL Milli-Q®-water was added and vortexed vigorously until DTT was solved.

•

Finally, 0.5 mL Milli-Q®-water was added and vortexed again for approximately 10 s.

DTT was always prepared freshly.

8.5.7 rea (8 M) Urea: M = 60.06 g mol–1; V = 1,0 mL; c = 8 M •

m = 8 mol L–1 * 0,001 L * 60.06 g mol–1 → 480.48 mg

•

480.48 mg urea were added into a 2.0 mL tube.

•

1 mL Milli-Q®-water was added and vortexed vigorously until urea was solved.

Urea was always prepared freshly as it develops a significant concentration of cyanate ions over time when it is kept at RT (Hinneburg et al. 2017).

8.5.8 Stain Solution 0.1% (w/v) Coomassie® Brilliant Blue R 205 in 50% methanol, 7% acetic acid. •

100 mL methanol, 86 mL Milli-Q®-water and 14 mL acetic acid were added into a 250 mL Schott/Duran flask.

•

100 mL x 0.79 g cm–3 + 86 mL x 1.0 g cm–3 + 14 mL x 1.05 g cm–3 = 179.7 g

•

179.7 g x 0.001 = 0.1797 g Coomassie® Brilliant Blue R 205 in 200 mL solution.

Stain solution was stored at RT until usage. xxxiii

Appendix

8.5.9 Destain Solutions Destain solution I: •

50% methanol, 7% acetic acid.

•

100 mL methanol, 86 mL Milli-Q®-water and 14 mL acetic acid were added into a 250 mL Schott/Duran flask.

Destain solution II: •

90% methanol, 10% acetic acid.

•

180 mL methanol, and 20 mL acetic acid were added into a 250 mL Schott/Duran flask.

Both destain solutions were stored at RT until usage.

8.5.10 Polyvinylpyrrolidone 40 0.1% (w/v) 0.1% PVP40 in 50 % methanol (v/v) •

10 mL x 0.79 g cm–3 + 10 mL x 1.0 g cm–3 = 17.9 g

•

179 g x 0.001 = 0.0179 g PVP40

•

17.9 mg PVP40 was transferred into a measuring cylinder (50 mL).

•

50% methanol was added until a volume of 20 mL was reached.

•

0.1% PVP40 solution was transferred into a 50 mL Falcon tube.

PVP40 solution was stored at RT.

8.5.11 2-Aminobenzamide Label Solvent mixture: •

500 µL DMSO and 150 µL acetic acid were mixed together in a 1.5 mL tube.

•

Tube was sonicated in a ultrasonic bath for 3 min by using a floating tube rack.

Labeling reagent 2AB: M = 136.12 g mol−1; V = 0.00065 L; c = 0.35 M •

m = 0.35 mol L−1 x 136.12 g mol−1 x 0.00065 L = 30.97mg

•

30.97 mg 2AB was transferred into a 1.5 mL tube.

•

650 µL of solvent mixture was added to 2AB.

•

Tube was sonicated in ultrasonic bath for 3 min using a floating tube rack.

Reducing reagent NaBH3CN: M = 62.84 g mol−1; V = 0.00065 L; c = 1.0 M •

m = 1 mol L−1 x 62.84 g mol−1 x 0.00065 L x 1.0 M = 40.85 mg

•

40.84 mg NaBH3CN was transferred into a 1.5 mL tube.

•

650 µL of solvent mixture containing labeling reagent was added to NaBH3CN.

•

Tube was sonicated in ultrasonic bath for 3 min using a floating tube rack.

Prepared 2AB label was stored up to six months at −20°C until usage (Wuhrer et al. 2009b).

xxxiv

Appendix

8.5.12 Ammonium Formate (50 mM, pH 4.4) NH4HCO2: M = 63.06 g mol−1; V = 2.0 L; c = 0.05 M •

m = 0.05 mol L−1 x 63.06 g mol−1 x 2.0 L = 1.803 g

•

3.606 g NH4HCO2 was transferred into a measuring cylinder (1 L).

•

Milli-Q®-water was added until a volume of 1 L was reached.

•

Solvent mixture was transferred into a 1 L Schott/Duran flask and stirred on a stirrer magnet until NH4HCO2 was completely dissolved.

•

pH was adjusted with 25% HCl (approximately 6 mL were needed).

•

For solvent A: 20% (200 mL) NH4HCO2) was added to 80% (800 mL) ACN in a second 1 L Schott/Duran flask.

•

For solvent B: 80% (800 mL) NH4HCO2) was added to 20% (200 mL) ACN in a third 1 L Schott/Duran flask.

8.5.13 Ammonium Acetate (5 mM, pH 6.4) NH4CH3CO2: M = 77.08 g mol−1; V = 1.0 L; c = 0.005 M •

m = 0.005 mol L−1 x 77.08 g mol−1 x 1.0 L = 0.3854 g

•

0.3854 g NH4CH3CO2 was transferred into a measuring cylinder.

•

Milli-Q®-water was added until a volume of 1 L was reached.

•

Solvent mixture was transferred into a 1 L Schott/Duran flask and stirred on a stirrer magnet until NH4CH3CO2 was completely dissolved.

•

pH did not need to be adjusted as 6.4 is its natural value.

xxxv