Dr Osama Chahrour *, Diego Cobice, Dr John Malone, Dr Dan Fletcher, Dr Alan Thompson and Cliodhna McGurgan. *Presenting author. Almac Sciences, 20 ...
5 Dimensional Structural Characterization of Synthetic Peptides Dr Osama Chahrour *, Diego Cobice, Dr John Malone, Dr Dan Fletcher, Dr Alan Thompson and Cliodhna McGurgan *Presenting author. Almac Sciences, 20 Seagoe Industrial Estate, Craigavon, Northern Ireland, BT63 5QD
Background There is currently growing interest in the therapeutic benefits of synthetic peptides. These molecules, like small molecule pharmaceuticals, require comprehensive characterization to unequivocally confirm the structure as per the USP guidelines. Full identity elucidation requires integration of orthogonal techniques to overcome the limitations associated with each technique. Presented is an example of the characterization protocol developed at Almac for synthetic peptides. This process confirms peptide sequence by Nuclear Magnetic Resonance and Tandem Mass Spectrometry, peptide molecular formula by accurate mass determination, chiral purity by pre-column derivatization of the peptide hydrolysates followed by liquid chromatography with mass spectrometry detection as well as ratio determination of each amino acid in the sequence. This characterization protocol goes beyond the requirements of the USP and gives confidence in the identification of synthetic peptides. The protocol has already been used for peptides containing up to 34 amino acids . A case study is presented herein.
Peptide Sequencing by NMR ~25 mg of a 20-mer peptide was dissolved in DMSO-d6. Spectra were obtained using a Bruker Avance NMR spectrometer operating at 500 MHz running under TopSpin 2.1. The sample was run at 28 and 50°C in the presence and absence of TFA. 1H and 13C spectra were assigned using DEPT-135, HSQC, HMBC, ROESY and TOCSY experiments. Individual peaks were picked out in the HSQC (finger-printing) and their multiplicity determined with the aid of the DEPT-135 spectrum. The correct number and multiplicity of carbons and protons was also confirmed. COSY-DQF, Transverse-ROESY and HSQC-TOCSY data was to assign the protons to individual amino acid side chains and confirm they agreed with the expected residues. The ROESY and HMBC data was used to determine the relative positions and sequence of amino acids within the molecule
Peptide Sequencing by LC-ESI-QTOF
Chirality Determination by HPLC-MS
The peptide precursor ions identified during the accurate mass determination were fragmented in the tandem mass spectrometer and the product (fragment) ions were subsequently mass analysed. The implemented low energy collision induced dissociation (CID) primarily produced y and b type ions. The data were processed using the Molecular Feature Extractor algorithm of MassHunter and matched with theoretical peptide fragment ions. The majority of y and b ions observed were within 10 ppm difference.
The chiral purity of the amino acids in the peptide were determined by acid hydrolysis followed by amino acid separation. Racemisation was circumvented by hydrolysing peptides in DCI/ [D4] acetic acid (1:1). Amino acids were then derivatised with Marfey's reagent and analysed by LC-MS. This method allows the original chiral purity of each amino acid in a peptide to be determined using common achiral reversed-phase HPLC columns. Use of ESI prevented fragmentation during ionisation and allowed the protonated molecular ion to be monitored for each amino acid.
ppm
3.7
G2-G3 G7-R2
Figure 5: Labelled Hydrolysis
3.8
3.9
4.0
4.1
R4-G16 R5-K20
4.2
4.3
Figure 6: Marfey Reaction
4.4
Opera tor: M alone , Joh n Print ing Ti me: 1 0:23: 47 Tue, May 27 , 201 4
8.30
8.25
8.20
8.15
8.10
8.05
8.00
7.95
7.90
7.85
7.80
7.75
7.70
7.65
7.60
7.55
7.50
7.45
4.5 ppm
X IC
Figure 3: Isotopic distribution matching between actual spectrum (black line) and FBF calculated theoretical distribution (red boxes)
+Q 1
io n s ) :
3 8 4 .2
amu
fro m
S a m p le
ID Source
Formula
Diff (ppm)
TRUE
FBF
C79H133N23O27
-1.60
Figure 2: Isotopic distribution matching between actual spectrum (black line) and FBF calculated theoretical distribution (red boxes) and table of mass confirmation.
(K
2 6 MA Y20 1 4A 0 0 04 )
of
K
1 2:19 M onday , May 26, 20 1 4 I nst I .D. E U - 0 0 1 27
2 6 M ...
M a x.
2 .3 e 6
cp s.
L-Isoleucine L-Leucine
1 .8 e 6 1 .6 e 6 1 .4 e 6 1 .2 e 6 1 .0 e 6 8 .0 e 5
D-Leucine
D-Isoleucine
6 .0 e 5 4 .0 e 5 3 1 .7 2 3 1 .4 0
2 .0 e 5
X IC
Precursor ion
m/z (prec.)
of
+Q 1
2 7 .5 MI
(1 9
2 8 .0 io n s ) :
4 1 8 .2
2 8 .5 amu
S a m p le
2 9 .5 4
(K
3 0 .0 3 0 .5 T im e , m in 2 6 MA Y20 1 4A 0 0 04 ) o f
3 1 .0 K
3 1 .5
3 2 .0
3 2 .5
2 6 M ...
M a x.
3 3 .0 3 .3 e 6
cp s.
2 8 .8 7
3 .3 e 6 3 .2 e 6
Number of charges Z
2 9 .0
fro m
3 .0 e 6 2 .8 e 6
L-Phenylalanine
2 .6 e 6 2 .4 e 6
(M+H)+ Product Ion
18X6.9849
1
2 .2 e 6 2 .0 e 6 1 .8 e 6 1 .6 e 6 1 .4 e 6
D-
Phenylalanine
1 .2 e 6
m/z
m/z (prod.)
Diff (ppm)
Sequence
Z
1 .0 e 6 8 .0 e 5 6 .0 e 5
3 0 .8 7
4 .0 e 5 2 .0 e 5 0 .0 2 7 .0
2 7 .5
2 8 .0
2 8 .5
2 9 .0
Page
Best
4
T ime: D ate: 3 65_K
2 9 .1 6
2 .0 e 6
Intensit y, cps
The accurate mass of the peptide was determined using an Agilent LC-QTOF to within 2ppm of theoretical. The Find By formula Algorithm of the MassHunter software was used to map the MS(1) data. It identified all major cluster ions and the algorithm confirmed the empirical formula.
(1 9
A cq. A cq. * API
2 8 .6 5
0 .0 2 7 .0
Accurate Mass Determination by LC-ESI-TOF
MI
2 .3 e 6 2 .2 e 6
Intensit y, cps
Figure 1: Intra-residue NH-H COSY correlations (green/blue). ROESY Inter-residue correlations (red) across the amide bonds from the -H of residue n to the NH of the residue n+1 were used to confirm the correct peptide sequence .
of
Acq. File : K 2 6MAY20 14A.w iff Batc h Nam e: K 26MAY2 014A. dab Coll ected by: \jma lone@ pharm s-serv ices. com
1
of
2 9 .5
1
3 0 .0 T im e , m in
3 0 .5
3 1 .0
3 1 .5
* Almac Scien ces 0 641E0 002C
3 2 .0
3 2 .5
P rojec t
3 3 .0
No.
b6
3X7.1777
3X7.183
-13.3
GGGR1GA
b10 y5 y6 y7 y8
78X.3853 5X2.3074 6X1.3497 7X4.431 8X5.4695
78X.3791 5X2.3089 6X1.3515 7X4.4356 8X5.4727
7.8 -2.9 -2.8 -5.9 -3.8
GGGR1GAGR2LQ GSLR5K R4GSLR5K LR4GSLR5K ALR4GSLR5K
1 1 1 1 1
y10
1X55.6084
1X55.6095
-1.1
R3LALR4GSLR5K
1
Amino acid analysis was carried out by acid, oxidative and alkaline hydrolysis. The individual amino acids were then analysed with HPLC-UV/FLU and/or Biochrom amino acid analysers using classical ion-exchange liquid chromatography with post-column Ninhydrin derivatisation and photometric detection. Li-Citrate as well as Na-Citrate buffer system elution was used to cover all the amino acids. The presence and correct number of amino acids in the peptide sequence were confirmed.
y11
1X83.6594
1X83.6681
-7.4
QR3LALR4GSLR5K
1
Conclusion
1
b10-H2O
7X4.3609
7X4.3686
-10
GGGR1GAGR2LQ
1
y11-NH3
1X66.648
1X66.6416
5.5
QR3LALR4GSLR5K
1
Figure 7: Example representative L and D amino acid standard chromatograms
Amino Acid Analysis
A summary of the results is given in the table below: G G G R1 G A G R2 L Q R3 L A L R4 G Sequence Confirmed by NMR
S
L
R5 K
Sequence confirmed by CID-Hybrid MS Chirality Confirmed by LC-MS Figure 4: Example of peptide precursor ion and corresponding product ions
The protocol for peptide characterization has been fully implemented by Almac has been used routinely to unequivocally confirm the structure of an number of peptides for our clients.
