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Book Peirce&Wait 9781603273091 Proof1 October 16, 2008

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Chapter 8

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Enrichment of Brain Plasma Membranes by Affinity Two-Phase Partitioning

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Jens Schindler and Hans Gerd Nothwang

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Plasma membranes encompass a complex and varying set of proteins essential to life. In addition, plasma membrane proteins represent the majority of all known drug targets. The characterization of plasma membrane proteomes is, therefore, of eminent importance. A current bottleneck is the lack of efficient protocols to isolate plasma membranes from tissues or entire organs. To this end, we recently established a simple and effective isolation procedure which is based on aqueous polymer two-phase systems. In this chapter, we provide a detailed protocol for the isolation of plasma membranes from brain tissue, which could easily be adapted to other sources.

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Abstract

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Key words: Brain, plasma membrane, affinity partitioning, two-phase system, enrichment.

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1. Introduction

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Plasma membranes (PMs) represent the interface between a cell and its environment and thus are central to many physiological processes such as cell-cell interactions, signal transduction, and molecular transport. Their importance is stressed by the fact that PM proteins account for approximately 70% of all known drug targets (1). In the brain, many PM proteins determine neuronal function, and their characterization, therefore, is essential for a better understanding of brain structure and neuronal processing. However, the analysis of this class of low abundant proteins at the high anatomical resolution required in heterogenous tissues such as the mammalian brain necessites the application of selective and efficient protocols: selectivity to reduce the contamination of the low abundant PM proteins by proteins from different

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Matthew J. Peirce, Robin Wait (eds.), Membrane Proteomics: Methods and Protocols, vol. 528 C 2009 Humana Press, a part of Springer Science+Business Media DOI 10.1007/978-1-60327-310-7 8 Springerprotocols.com

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Schindler and Nothwang

origin; efficiency to reduce the losses of PM proteins during fractionation. One method to fulfill these criteria is affinity partitioning in aqueous two-phase systems. Aqueous two-phase systems form when solutions of two polymers are mixed above a critical concentration. Each of the resulting phases will be enriched in one of the two polymers (2). In the two-phase systems, membranes differing in their subcellular origin partition preferentially in either of the two phases. This can be exploited for the efficient enrichment of a specific subcellular membrane compartment such as the PM. In a dextran/polyethylene glycol (PEG) two-phase system, for instance, PMs show the highest affinity of all subcellular membranes to partition in the top phase enriched in PEG (2). In addition to the polymer composition, partitioning behavior of the membranes can be altered by salt concentrations in the twophase system, by the polymer concentrations, and by the presence of affinity ligands such as wheat germ agglutinin (WGA) (3–5). Each of these factors has to be adjusted to the tissue and subcellular compartment of interest. The protocol we describe here is used to enrich brain plasma membranes from a crude membrane fraction by affinity two-phase partitioning (6). First, microsomes containing PMs, are isolated by differential centrifugation (7). In a second step, PMs are further enriched in a two-phase system of PEG and dextran. The use of the affinity ligand WGA in the following step results in the yield of highly purified PMs. In a final step, integral PM proteins are separated from peripheral PM proteins by high-salt and high-pH washes (8, 9). This enables separate analysis of these two important classes of PM proteins.

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2. Materials

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Due to the strong influence of ions on membrane partitioning in the two-phase systems, double distilled water should be used throughout the experiments.

2.1. Preparation of Microsomes

1. Glass-Teflon homogenizer 2. Homogenization buffer: sucrose (250 mM), Tris (15 mM), complete protease inhibitor (Roche), pH 7.8

2.2. Preparation of WGA-Dextran

1. 2. 3. 4. 5. 6.

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2.2.1. Activation of Dextran

Dimethyl sulfoxide (DMSO) (see Note 1) Triethylamine (see Note 1) Dichloromethane (see Note 1) Dextran T500 (see Note 2) Tresyl chloride Dialysis tubes (MWCO 12,000-14,000) (Roth, Karlsruhe, Gemany)


Enrichment of Brain Plasma Membranes

2.2.2. Coupling of WGA

1. Coupling buffer: NaCl (500 mM), NaH2 PO4 (100 mM), pH 7.5 2. Quenching buffer: Tris-HCl (400 mM), pH 7.5 3. Jumbosep centrifugal device (MWCO 100,000) (VWR)

2.3. Affinity of Two-Phase Partitioning

1. Dextran stock solution: Dextran T500 (20% w/w) (see Note 2) 2. PEG stock solution: PEG 3350 (40% w/w) 3. Tris-H2 SO4 : Tris (200 mM), pH 7.8 adjusted with H2 SO4 4. Borate buffer: boric acid (200 mM), pH 7.8 adjusted with Tris 5. Li2 SO4 stock solution: Li2 SO4 (200 mM) 6. WGA-Dextran 7. N-acetyl-D-glucosamine solution: N-acetyl-D-glucosamine (100 mM), sucrose (250 mM), Tris (5 mM), pH 7.8

2.4. High-Salt and High-pH Washing

1. Glass-Teflon homogenizer 2. High-salt buffer: KCl (1 M), Tris (15 mM), pH 7.4 3. High-pH buffer: Na2 CO3 (100 mM) (see Note 3)

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3. Methods

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3.1. Preparation of Microsomes

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1. Homogenize 1.5 g of brain tissue in 4.5 mL of homogenization buffer by 20 strokes at 250 rpm in the glass-Teflon homogenizer. For smaller amounts of brain tissue, adjust the amount of homogenization buffer accordingly. 2. Centrifuge (3,000 × g) for 10 min at 4◦ C to sediment nuclei and cell debris. 3. Remove the supernatant and store on ice until further use. 4. Re-extract the pellet with a pipette in the same volume of homogenization buffer, as used for initial homogenization. 5. Centrifuge (3,000 × g) for 10 min at 4◦ C. 6. Repeat Steps 3–5. 7. Combine all three supernatants and centrifuge (10,000 × g) for 12 min at 4◦ C to sediment mitochondria. 8. Remove the supernatant and store on ice until further use. 9. Re-extract the pellet with a pipette in the same volume of homogenization buffer as removed before. 10. Centrifuge (10,000 × g) for 12 min at 4◦ C. 11. Repeat Steps 8–10. 12. Combine the three supernatants of the 10,000 × g centrifugation steps and centrifuge (100,000 × g) for 1 h at 4◦ C. 13. Discard the supernatant. The resulting pellet represents the microsomes that are further purified by affinity two-phase partitioning. The pellet can be stored at −80◦ C.

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3.2. Preparation of WGA-Dextran

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3.2.1. Activation of Dextran

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3.2.2. Coupling of WGA

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1. Dissolve 2 g of tresyl-dextran in 10 mL coupling buffer in a glass tube and 10 mg WGA in 1 ml coupling buffer. 2. Add the WGA-solution drop wise with a pipette to the tresyl-dextran solution with vigorous vortexing (approximately 10 min). Incubate the mixture over night at 4◦ C with agitation. 3. Add 10 mL of quenching buffer to terminate the reaction and to inactivate unreacted tresyl groups. Incubate for 2 h at 4◦ C with agitation (see Note 7). 4. Add the mixture to Jumbosep centrifugal devices, fill up with distilled water to a final volume of 60 mL, and centrifuge until the volume has decreased to one third. Repeat the procedure five times (see Note 8). 5. Freeze-dry WGA-dextran (see Notes 2 and 6). Determine the coupling efficiency by Bradford assay (Pierce) using WGA as standard (see Note 9).

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1. Dissolve 5 g of freeze-dried Dextran T500 in 25 mL of DMSO at room temperature in a glass beaker dried in an oven at 100◦ C over night (see Notes 1 and 2). Dissolving dextran can take up to 1 h. 2. Add slowly 1 mL of triethylamine followed by 5 mL of dichloromethane within approximately 10 min under stirring to avoid precipitation of dextran (see Note 1). 3. Chill on ice while stirring (see Note 4). 4. Add slowly 0.35 g (220 μL) of tresyl chloride under vigorous stirring on ice. Stir on ice for 1 h. 5. Stir the solution at room temperature over night. 6. Add 50 mL of dichloromethane to precipitate dextran (see Note 5). 7. Wash four times with 25 mL dichloromethane while kneading the precipitate with a glass rod until firm consistency (see Note 5). 8. Dissolve the washed precipitate in 30 mL water and dialyze against distilled water until the dialysis tube contains only one clear solution. 9. Freeze-dry tresyl-dextran and store at −20◦ C (see Notes 2 and 6).

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3.3. Affinity Two-Phase Partitioning

All steps of the affinity two-phase partitioning protocol have to be performed at 4◦ C. Working at room temperature prevents phase separation. The procedure is illustrated in Fig. 8.1. The numbers in Fig. 8.1 correspond to the numbered two-phase systems given in Table 8.1, and the letters refer to the phases as indicated in the protocol given below. 1. Prepare all two-phase systems with the compositions indicated in Table 8.1 one day prior to use. Mix them by



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Fig. 8.1. Schematic illustration of the affinity partitioning procedure.

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Table 8.1 Composition of two-phase systems

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Two-phase system 2 3 4

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Dextran stock solution

1.26 g

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PEG stock solution

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0.63 g

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Tris-H2 SO4

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Borate-buffer

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Li2 SO4

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0.04 g

1.81 g

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WGA-dextran

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Water am

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1.81 g

WGA-dextran = 800 μg/(coupling degree [μg/mg dextran]×1,000) (see Note 10) bm dextran = (0. 504 g–mWGA-dextran ) × 5 (see Note 10) cm water = 8 g − (mdextran + mWGA−dextran + mPEG + mboratebuffer + mLi2 SO4 ) (see Note 10)



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20 invertations, vortexing for 10 s, another 20 invertations, and store the mixtures at 4◦ C over night. Two-phase systems with the top phase enriched in PEG and the bottom phase enriched in dextran will form over night. Add 400 μL of microsomes resuspended in homogenization buffer to the two-phase system 1. If less than 400 μL are used, make up to 400 μL with distilled water. Mix by 20 inversions, vortexing for 10 s, and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Remove the top phase (top phase A) (see Note 11) and store it at 4◦ C until further usage. Add a similar volume of fresh top phase from two-phase system 2. Mix by 20 inversions, vortexing for 10 s, and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Remove the top phase (top phase B) and combine it with top phase A. Layer the combined top phases A + B onto the bottom phase of two phase system 2. Mix by 20 inversions, vortexing for 10 s, and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Remove the resulting top phase C and mix it with fresh bottom phase from two-phase system 3 by 20 inversions, vortexing for 10 s and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Remove the resulting top phase D and mix it with fresh bottom phase from two-phase system 4 by 20 inversions, vortexing for 10 s, and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Discard the top phase E and mix the bottom phase with a similar volume of fresh top phase from two-phase system 5 with 20 inversions, vortexing for 10 s, and another 20 inversions. Accelerate phase separation by centrifugation for 5 min at 150 × g. Remove the resulting top phase F . Dilute the bottom phase with a 10-fold volume of N-acetyl-D-glucosamine solution and centrifuge at 100,000 × g for 90 min. This will release the PMs from WGA-dextran and result in their sedimentation.

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3.4. High-Salt and High-pH Washing to Enrich for Integral Membrane Proteins

To enrich integral PM proteins, a high-salt and high-pH wash is recommended. Only peripheral membrane proteins will be solubilized in the washing buffers. 1. Resuspend the final pellet obtained in the affinity partitioning procedure in ice- cold high-salt buffer with a glass-Teflon homogenizer and centrifuge for 1 h at 233,000 × g. Repeat this step twice. Each time, the supernatant, representing peripheral PM proteins, is collected. 2. Resuspend the pellet obtained in Step 1 in ice-cold high-pH buffer with a glass-Teflon homogenizer and centrifuge for 1 h at 233,000 × g. Repeat this step twice. Collect each time the supernatant, which represents peripheral PM proteins.



3. Combine all six supernatants, enriched in peripheral PM proteins and store them as well as the pellet, enriched in integral membrane proteins, at −80◦ C.

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4. Notes

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1. DMSO, triethylamine and dichloromethane are dried with molecular sieves prior to use. For that purpose, dry an Erlenmeyer flask containing 0.02 g of molecular sieves (Sigma) for each milliliter solvent used next day in an oven at 100◦ C over night for each solvent,. The next day, cool down to room temperature, and add the desired amount of DMSO, triethylamine, or dichloromethane to the molecular sieves. Close the flasks tightly with parafilm and leave them at room temperature for at least 5 h. 2. Dextran can contain up to 10% water and for that reason has to be freeze-dried. For freeze-drying, dissolve dextran in distilled water in a plastic dish with a large surface (e.g., Petri dish), freeze it at −80◦ C and dry it by sublimating the water under vacuum. Store the freeze-dried dextran in closed plastic tubes sealed tightly with parafilm at −20◦ C. Let it come to room temperature before opening in order to protect it from humidity. 3. pH has to be 11, less alkaline pH results in less elimination of peripheral membrane proteins. 4. Cooling the solution on ice usually increases the viscosity of the solution and small amounts of dextran might precipitate. If this happens, try to stir as well as possible. After half an hour, stirring should be no problem. 5. When precipitating the dextran in Step 6, it is clear and slimy. For washing and kneading, press this slime against the beaker with the glass rod. After washing, the product should be white and crystalline. 6. Freeze dried tresyl-dextran and WGA-dextran can be stored for several months at −20◦ C. Take care that tresyl-dextran and WGA-dextran do not make contact with water. 7. Tresyl groups react with the amino groups of Tris in the quenching buffer. The termination of the reaction is essential to avoid reactions with any amino group in the subsequent two-phase partitioning procedure. 8. Proper washing of WGA-coupled dextran is essential to remove uncoupled WGA and the salt of the buffers used for the coupling procedure. Salts alter the partitioning of membranes in the two-phase systems. 9. It is essential to know precisely the extent of coupling of WGA per mg dextran, as defined amounts of WGA (in the form of WGA-dextran) are used in the affinity partitioning protocol.

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10. A two-phase system of 8 g with a final concentration of 6.3% of dextran and 800 μg WGA is required. As in this step, WGA is used coupled to dextran (WGA-dextran), and the amount of WGA coupled to dextran might vary between different coupling experiments, the amount of WGA-dextran, dextran, and water in the two-phase system have to be calculated. a) 800 μg of WGA in the form of WGA-dextran are required. With a degree of coupling of 4 μg WGA/mg dextran, 0.2 g WGA-dextran is used. b) A total of 0.504 g of dextran is required to get a final concentration of 6.3% in an 8 g system. As already a certain amount of dextran is present in the form of WGA-dextran, this amount has to be subtracted. This results in 0.304 g dextran in our example (0.504–0.2 g). Using a stock solution of 20%, 5 × 0.304 g (i.e., 1.52 g) of this stock solution has to be used to end up with 0.304 g dextran. c) The two-phase system is adjusted to 8 g using water. Hence, the amount of all other ingredients has to be subtracted to calculate how much water is missing. 11. In the two-phase systems, interphases are always assigned to the bottom phase.

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Acknowledgements

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This work was funded in part by the Nano+Bio-Center of the University of Kaiserslautern.

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References

1. Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1, 727–730. 2. Albertsson P-A (1971) Binding Studies. In: Partition of cell particles and macromolecules (Albertsson P.-A., ed), pp. 233–242. New York: Wiley-Interscience. 3. Gierow P, Sommarin M, Larsson C, Jergil B (1986) Fractionation of rat liver plasmamembrane regions by two-phase partitioning. Biochem J 235, 685–691. 4. Persson A, Johansson B, Olsson H, Jergil B (1991) Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning. Biochem J 273, 173–177. 5. Persson A, Jergil B (1995) The purification of membranes by affinity partitioning. FASEB J 9, 1304–1310.

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6. Schindler J, Lewandrowski U, Sickmann A, Friauf E, Gerd NH (2006) Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning. Mol Cell Proteomics 5, 390–400. 7. Aronson NN, Jr., Touster O (1974) Isolation of rat liver plasma membrane fragments in isotonic sucrose. Methods Enzymol 31, 90–102. 8. Fujiki Y, Hubbard AL, Fowler S, Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93, 97–102. 9. Taylor RS, Wu CC, Hays LG, Eng JK, Yates JR, Howell KE (2000) Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation. Electrophoresis 21, 3441–3459.


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