Microsomal Proteomics

Summary

Proteomic profiling of subcellular compartments has many advantages over traditional proteomic approaches using whole cell lysates as it allows for detailed proteome analysis of a specific organelle and corresponding functional characteristics. The microsome is a critical, membranous compartment involved in the synthesis, sorting, and secretion of proteins as well as other metabolic functions. This chapter will describe detailed methods for the isolation of microsomal organelles including the ER, Golgi, and prechylomicron transport vesicle (PCTV), a recently identified vesicular system involved in intestinal lipoprotein assembly and secretion. Particular focus is given to the isolation of microsomes from primary hepatocytes and enterocytes freshly isolated from rodent liver and intestinal tissue, and their proteomic profiling using a combination of two-dimensional gel electrophoresis and mass spectrometry.

17.1 Introduction

Proteomics involves the integration of technologies to analyse the complete complement of proteins expressed (referred to as the proteome) by a biological system in response to stimuli under different physiological/pathological conditions. Examining changes in the proteome offers insight into understanding cellular and molecular mechanisms that cannot be obtained through genomic analysis. The information gap between a genome and associated gene products is largely attributed to post-translational modifications. These modifications have been shown to modulate pivotal regulatory processes such as protein turnover, protein activity, and protein localization within a cell. In addition, virtually all known cellular signalling pathways are mediated through a complex cascade of reversible protein phosphorylation. The proteome is a dynamic feature, subject to changes due to developmental stage, disease state, or environmental conditions.

Currently, proteomic protocols commonly incorporate two-dimensional electrophoresis (2DE) for protein separation, where proteins are detected with various stains, and the protein profile is analysed using 2DE gel imaging software. Proteins of interest are identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) or tandem mass spectrometry (MS/MS). An important goal of proteomics research is to deposit the results into the proteome database, which contains both protein identification and corresponding functional characteristics. However, 2DE is not an effective technique for profiling membrane proteins because they are not easily solubilized for isoelectric focusing (IEF) (1), and they have difficulty entering the gel due to their size and hydrophobicity (2, 3). (see also Chapter “Difficult Proteins”). An alternative approach to identify membrane proteins is to digest in-solution and then apply to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tandem mass spectra of the peptides can be searched in databases to identify amino acid sequences (4, 5). In addition, 1-dimensional blue native polyacrylamide gel electrophoresis (1-D BN-PAGE; see Chapter 15) is an in-gel technique used to study membrane proteins. Coomassie dye in the sample buffer induces a charge shift in the membrane proteins. Aminocaproic acid also helps in their solubilization (6).

Current research using proteomics is focused on two main areas: expression proteomics, which measures the fluctuation of a protein level under certain conditions; and functional proteomics, which characterizes proteins in organelles and complexes. Expression proteomics involves investigating proteins altered in a disease or drug-treated state, compared to normal, to discover diagnostic markers or therapeutic targets (7). However, studies of whole proteomes are not representative of all the proteins contained in the cell since low-abundance proteins are not easily identified (8). A common approach used to enrich low-abundance proteins is to isolate individual subcellular compartments (see Chapters “Organelle Proteomics,” “Preparation and Analysis of Plastid Proteomes by 2DE” and “2DE for Proteome Analysis of Human Metaphase Chromosomes”). Using subcellular fractionation methods, individual organelles can be isolated and their protein complements resolved by proteomics. This method is particularly useful in the investigation of specific subcellular organelle(s) thought to be affected in a disease state. Morand et al.used this comparative approach to identify proteins in hepatic endoplasmic reticulum (ER) that were altered in nutritionally induced insulin resistance (9).

Microsomes are among the cell’s most active membranous structures involved in the synthesis, sorting, and secretion of proteins as well as other metabolic functions. The microsome consists of a complex network of continuous membranes including ER, ER-Golgi intermediate complex (ERGIC) – also referred to as the vesiculotubular clusters or pre-Golgi intermediates – and the Golgi apparatus. These organelles are membrane-bound compartments that have distinct functions. The trafficking of proteins and lipids in the microsome is mediated by vesicles that bud from the ER and fuse with the cis-Golgi compartment. COPII-coated and COPI-coated vesicles transport proteins from the ER to the Golgi, where the COPI-coated vesicles are also capable of retrograde movement (10). The ERGIC, a membrane system between rough ER and Golgi, is a transient cargo holder of vesicles (11, 12). COPII-coated vesicles also play an important role in anterograde movement from the ER to the ERGIC, and COPI-coated vesicles play a well-established role in retrograde traffic from the Golgi to ERGIC (13, 14). Studies in the small intestine have recently identified a unique intermediate vesicle compartment, prechylomicron transport vesicles (PCTVs), involved in intestinal packaging of absorbed lipids and formation of chylomicrons (15, 16). Recent proteomic analysis of the microsomal preparations has helped identify many unique microsome-associated proteins. Specifically, 141 proteins have been identified in the ER (17), 24 proteins have been identified in the ER-GIC (18), and over 400 proteins have been identified in the Golgi (19, 20). The ER and Golgi compartments are in a dynamic equilibrium involving continuous formation and fusion of secretory vesicles in both antegrade and retrograde directions. This results in a continuous change of molecular composition and makes pure isolation of distinct microsomal fractions very challenging. There are several approaches to examine the purity of organelle preparations. A simple way is to immunoblot for proteins which could easily contaminate. For example, when working with ER, is it common to immunoblot for Golgi and lysosomal protein markers. ‘Subtractive proteomics’ is a more advanced technique, which compares a specific cellular compartment of interest to a ‘background’ complex (21). In 2003, Schirmer et al. verified proteins in the nuclear envelope by subtracting proteins also found in the microsomal membrane (22).

The following chapter will describe proteomic techniques used to study microsomal fractions. We will focus on microsomal proteomics in primary cultured hepatocytes and enterocytes. Techniques for primary cell isolation, subcellular fractionation, 2DE, and MS will be described. In addition, we describe the methodology for isolation and proteomic profiling of the recently identified microsomal compartment, PCTV.

17.2 Materials

Microsomal proteomic analysis can be performed on primary cells from any animal model. Here, male Syrian golden hamsters (Mesocricetus auratus) (Charles River, Montreal, QC, Canada) maintained on a chow diet (Dyets Inc, Bethlehem, PA) are used. An overview of the entire procedure is given in Fig. 1.

Fig. 1.

A schematic overview of the protocols used to perform proteomic analysis of microsomal fractions. Following isolation of primary cells from rodent tissue, the microsomal fraction can be analysed by 2DE followed by MS, or organellar extract subjected to direct trypsin digestion followed by direct injection of the peptide mixture into the LC-MS/MS. Database searching can identify the proteins of interest.

17.2.1 Animal Surgery

• 1. Anaesthesia (isofluorane, nitrous oxide, and oxygen, Baxter, Toronto, ON) and chamber.

• 2. Surgical tools.

17.2.1.1 Primary Cell Isolation: Hepatocytes

• 1. Liver perfusion, digest, wash media (Life Technologies, Gaithersburg, MO).

• 2. Peristaltic pump (Amersham Biosciences, Piscataway, NJ).

• 3. Scissors, cell strainer.

• 4. 1× PBS

17.2.1.2 Primary Cell Isolation: Enterocytes

• 1. 100-mm Petridish, 18-gauge needle and syringe.

• 2. 1× PBS.

• 3. Cell Recovery Solution (BD Biosciences) (see Note 1 ).

• 4. Orbital shaker.

17.2.2 Microsome Isolation

• 1. Primary cells.

• 2. Buffer A (10 mM HEPES, pH 7.2, 0.25 M sucrose 2 mM EDTA) and protease inhibitor cocktail (PI) (Complete-Mini, EDTA-free, Roche, Mississauga, ON, Canada).

• 3. Homogenizer (Parr bomb + N₂ pressure) (Parr Instruments, Moline IL).

• 4. Ultracentrifuge (Beckman Optima LE-80, SW41Ti rotor, Mississauga, ON, Canada).

• 5. 12-mL ultracentrifuge tubes (Beckman).

17.2.2.1 Microsome Isolation for Prechylomicron Transport Vesicle Budding Assay

• 1. Buffer B (137 mM NaCl, 1.5 mM EDTA, 11.5 mM KH₂PO₄, 8 mM Na₂HPO₄, 2.2 mM KCl, 0.5 mM dithiothreitol (DTT), pH 7.2) supplemented with 10 mM glutamine.

• 2. 50 μCi [³H]-oleic acid (PerkinElmer, Boston, MA).

• 3. Bovine serum albumin (BSA) (Sigma Aldrich, Oakville, ON, Canada).

• 4. Sodium oleate (Sigma Aldrich).

• 5. 1× PBS.

17.2.2.1.1 Cytosol Preparation

• 1. Buffer C (25 mM HEPES, pH 7.2, 125 mM KCl, 2.5 mM MgCl₂, 2 mM DTT, 0.5 mM EDTA) and PI.

• 2. Amicon Ultra 15 Centrifugal Filter Devices, 10,000 M_r cut-off (Millipore, Billerica, MA).

• 3. Anti-enolase antibody (Santa Cruz Biotechnology).

17.2.2.2 Endoplasmic Reticulum and Golgi Apparatus Isolations

• 1. 0.25 M, 0.86 M, 1.15 M, 1.22 M sucrose dissolved in 10 mM HEPES, pH 7.2.

• 2. Anti-calnexin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

• 3. Anti-GS28 antibody (Stressgen Bioreagents, Ann Arbor, MI).

17.2.2.2.1 PCTV Budding Assay

• 1. Cytosol buffer (25 mM HEPES, 125 mM KCl, 2.5 mM MgCl₂, 2.5 mM DTT, pH 7.2).

• 2. ATP mixture (5 mM ATP, 25 mM phosphocreatine, 25 units phosphokinase) in 10 mM HEPES, pH 7.2 (see Note 2 ).

• 3. 2.5 mM Mg²⁺, 2.5 mM Ca²⁺, 5 mM DTT.

• 4. E₆₀₀ prep (200 μL E₆₀₀ stock in 5 mL 10 mM HEPES, pH 7.2) (Sigma Aldrich).

• 5. Transport Buffer (30 mM HEPES, 0.25 M sucrose, 2.5 mM MgCl₂, 30 mM KCl, pH 7.2).

• 6. 0.10 M and 1.15 M sucrose in 10 mM HEPES, pH 7.2.

• 7. Gradient maker and stir bar.

17.2.3 Isoelectric Focusing

• 1. Lysis buffer (0.25 M sucrose, 10 mM Tris–HCl, pH 7.4) and PI.

• 2. Acetone.

• 3. Reagent 3 from ReadyPrep Sequential Extraction Kit (BioRad, Hercules, CA).

• 4. IPGphore IEF Unit (Amersham Biosciences, Piscataway, NJ).

• 5. 24-cm pH 3–10 NL Immobiline Drystrips (Amersham Biosciences).

• 6. Rehydration buffer (8 M urea, 0.5% CHAPS, 15 mM DTT, 0.2% Pharmalyte 3–10, 4–7, or 6–11) and bromophenol blue.

• 7. Immobiline Drystrip holder (Amersham Biosciences).

• 8. Drystrip Cover Fluid (Amersham Biosciences).

• 9. Equilibration buffer (6 M urea, 30% glycerol and 2% SDS in 0.05 M Tris–HCl buffer).

• 10. DTT, iodoacetamide.

17.2.4 -Dimensional Polyacrylamide Gel Electrophoresis

• 1. Ettan DALTsix large format PAGE tank (Amersham Biosciences).

• 2. Stacking gel. 0.5% Agarose made up in 1× running buffer.

• 3. Resolving gel. 8% sodium dodecyl sulphate (SDS) polyacrylamide gel.

  • (a) M Tris–HCl, pH 8.8, 0.4% SDS.

  • (b) 40% acrylamide/bis solution (29:1 with 3.3%C).

  • (c) N,N,N,N′-Tetramethyl-ethylenediamine (TEMED) (BioRad).

  • (d) Ammonium persulphate (APS): prepare 1% solution in water.

• 4. 1× Running buffer (25 mM Tris–HCl, 192 mM Glycine, 0.1% SDS, pH 8.5).

• 5. Prestained molecular weight markers (Fermentas, Burlington, ON, Canada).

• 6. Deep Purple Total Protein Stain (Amersham Biosciences).

• 7. Typhoon^™ 9400 Imager (Amersham Biosciences).

17.2.5 Mass Spectrometry Sample Preparation

• 1. Pipette tips, scalpel, syringe.

• 2. 30 mM ammonium bicarbonate/40% acetonitrile.

• 3. 50 mM ammonium bicarbonate/1 mM CaCl₂ solution.

• 4. 50% acetonitrile/1% trifluoroacetic acid (TFA).

• 5. 20% formic acid/15% 2-propanol/25% acetonitrile.

• 6. 80% acetonitrile.

• 7. 0.1% trifluoroacetic acid.

• 8. 0.8 M guanidine chloride/2.5% TFA.

• 9. SpeedVac centrifuge (Savant, Tamsey, MA).

• 10. Sequencing-grade trypsin (Promega, Madison, WI) in 50 mM acetic acid.

• 11. C₁₈ ZipTip^™ (μZT) pipette tips (Millipore).

• 12. α-cyano-4-hydroxycinnamic acid (CHCA), 10 mg/mL.

• 13. Micromass MALDI-Q-ToF (Waters, Milford, MS).

17.2.6 Liquid Chromatography Tandem Mass Spectrometry Sample Preparation

• 1. Digestion buffer. 50 mM NH₄HCO₃.

• 2. 6 M urea/2 M thiourea solution. 6 M urea, 2 M thiourea, 10 mM Tris–HCl, 150 mM NaCl, 1 mM PMSF, pH 8.0 with protease inhibitor cocktail (PI).

• 3. 80 mM DTT stock solution.

• 4. 300 mM iodoacetamide stock solution.

• 5. LysC stock solution. To make 1 mL, dilute 0.5 μg in 1 mL digestion buffer. Separate into aliquots and store at −20°C.

• 6. Sequencing-grade trypsin (Promega).

• 7. Mobile phase (e.g. 10 mM ammonium acetate–methanol–acetonitrile (30:35:35)).

• 8. Thermo Electron LCQ Deca XP (Thermo Scientific, San Jose, CA) coupled with an Agilent capillary HPLC 1100 series (Agilent Technologies, Palo Alto, CA, USA) and C₁₈ column (150 mm × 4.6 mm, 5 μm, Zorbax Extend).

17.3 Methods

The proteomic methods described can be used on microsomes isolated from different tissues. In our laboratory we typically use hepatocytes or enterocytes from the Syrian golden hamster; however, this protocol should be applicable to other rodents. The procedures for microsome (Subheading 3.2 ), ER, and Golgi isolations (Subheading 3.2.1 ) have been adapted but significantly modified from a published protocol (23). The protocols for ER (Subheading 3.2.2 ) and cytosol (see “PCTV Budding Assay” in Subheading 3.2.2 ) isolations for PCTV budding assay, as well as PCTV budding assay (Subheading 3.2.2) have been previously published (24). An overview of cell fractionation can be seen in Fig. 2.

Fig. 2.

Protocols for preparation of total microsomal membranes and the PCTV budding assay. (a) Following the isolation of primary cells, centrifuge the homogenate at low speed in the ultracentrifuge to remove nuclei, mitochondria, and unbroken cells. Take the post-nuclear supernatant and spin at high speed to remove cytosol. Overlay the resulting microsomal pellet with a discontinuous sucrose gradient to isolate ER and Golgi. (b) Incubate ER with cytosol, ATP, and other factors to bud the PCTVs out of the ER. Overlay the PCTV mixture onto a continuous sucrose gradient and spin to purify the budded PCTVs.

17.3.1 Animal Surgery

• 1. Maintain male Syrian golden hamsters on a chow diet for at least 3 days prior to use and then fast overnight prior to killing.

• 2. Anaesthetize the animal with a continuous flow of the anaesthetic mixture.

• 3. Make an incision up the middle of the animal’s abdomen and prepare for liver perfusion.

17.3.1.1 Primary Cell Isolation: Hepatocytes

• 1. Insert a needle into the abdominal vena cava and secure with sutures.

• 2. Isolate the liver from the circulatory system by blocking the thoracic aorta, caudal vena cava, abdominal aorta, and abdominal vena cava with sutures.

• 3. Pump 50 mL of perfusion solution at 39°C into the liver using the peristaltic pump at the highest setting.

• 4. Snip the portal vein to allow perfusion media to escape the liver.

• 5. Pump 50 mL of liver digest medium at 39°C into the liver.

• 6. Cut out the liver and dice with scissors in wash media.

• 7. Pass the perfused liver through a cell strainer.

• 8. Wash the hepatocytes three times with 1× PBS to remove collagenase.

• 9. Centrifuge the hepatocytes at 100 × g to pellet.

17.3.1.2 Primary Cell Isolation: Enterocytes

• 1. Excise approximately 10 cm of the proximal end of the small intestine and place in a 100-mm dish of ice-cold 1× PBS.

• 2. Rinse the intestine several times with 1× PBS using an 18-gauge needle.

• 3. Cut the intestine longitudinally and subsequently cut into 1-cm fragments.

• 4. Immerse intestinal fragments in Cell Recovery Solution for 1 h at 4°C.

• 5. Wash the enterocytes using 1× PBS with agitation on an orbital shaker for 5 min.

• 6. On the shaker, add 5 mL of 1× PBS to the fragments and use fingers to tap intestine gently. At this time, villi will start dissociating from the intestinal fragments.

• 7. Collect the PBS with the suspended villi and repeat 4–5 times or until villi no longer dissociate from the intestinal fragments.

• 8. Pellet the suspended villi with a 3-min centrifugation at 200 × g.

17.3.2 Microsome Isolation

• 1. Resuspend the cell pellet in 10–20 mL cold buffer A with PI (see Note 3 ).

• 2. Homogenize the cells using a Parr bomb cell disruption vessel at 800 psi N₂ pressure for 35 min at 4°C.

• 3. Transfer the homogenate to a 12-mL ultracentrifuge tube and spin at 8,500 × g for 10 min at 4°C in an ultracentrifuge (Beckman Optima LE-80, SW41Ti rotor) to remove nuclei and mitochondria.

• 4. Take the post-nuclear supernatant (PNS) and centrifuge at 100,000 × g for 3 h at 4°C.

• 5. Discard the supernatant and retain microsome pellet.

17.3.2.1 Microsome Isolation for PCTV Budding Assay

• 1. Resuspend 20 × 10⁶ enterocytes in 20 mL cold buffer B supplemented with 10 mM glutamine and PI.

• 2. In a separate tube, add 50 μCi [³H]-oleic acid to 200 μL 10% BSA-10 mM oleate complex (see Note 4 ). Vortex and add to cells from step 1.

• 3. Incubate enterocytes at 37°C for 30 min with occasional mixing.

• 4. Centrifuge the cells at 1,000 × g and discard the supernatant.

• 5. Wash the pellet twice with 2% BSA in cold PBS.

• 6. Discard the supernatant and resuspend cells in 10–20 mL cold buffer A with PI.

• 7. Follow steps 2–5 from Subheading 3.2 .

17.3.2.1.1 Cytosol Preparation

• 1. Resuspend enterocytes in 10–20 mL cold buffer C with PI.

• 2. Homogenize the cells using a Parr bomb cell disruption vessel at 1,000 psi N₂ pressure for 40 min at 4°C.

• 3. Transfer the homogenate to a 12-mL ultracentrifuge tube and centrifuge at 8,500 × g for 10 min at 4°C in an ultracentrifuge (Beckman Optima LE-80, SW41Ti rotor).

• 4. Take the post-nuclear supernatant and centrifuge at 100,000 × g for 3 h at 4°C.

• 5. Take the supernatant (avoid lipid layer) and place in Amicon Ultra 15 Centrifugal tube.

• 6. Centrifuge at 4,000 × g until volume is less than 1 mL.

• 7. Add buffer C (no PI) and centrifuge until volume is less than 1 mL.

• 8. Repeat step 7 and concentrate until protein concentration in cytosol fraction is ∼20 mg/mL.

• 9. Prepare an equal volume cytosol and whole cell lysate as a positive control, for electrophoresis on 8% SDS-PAGE and transfer to PVDF membrane.

• 10. Immunoblot the membrane with anti-enolase antibody to determine if cytosol is present.

• 11. Immunoblot the membrane with anti-calnexin and/or anti-GS28 antibody to verify that cytosol is not contaminated with ER and/or cis-Golgi.

17.3.2.2 ER and Golgi Apparatus Isolations

• 1. Resuspend the microsome pellet in 3 mL of 1.22 M sucrose solution.

• 2. In the ultracentrifuge tube, carefully overlay the microsomal suspension with 2.6 mL each of 1.15 M, 0.86 M, and 0.25 M sucrose solutions.

• 3. Centrifuge the sucrose step gradient at 82,000 × g for 3 h at 4°C (SW41Ti rotor).

• 4. Take the pellet and 1.22 M sucrose layer as rough and smooth ER, respectively. The cis- and trans-Golgi membranes float at the 0.25/0.86 M and 0.86/1.15 M density interfaces, respectively (24). Remove by Pasteur pipette.

• 5. For highly purified ER, adjust the ER density to 1.22 M sucrose and repeat steps 2 and 3.

• 6. Fractionate by unloading 22 × 500 μL aliquots from the top of the tube.

• 7. Prepare equal volumes of each fraction, along with whole cell lysate as a positive control, for electrophoresis on 8% SDS-PAGE and transfer to PVDF membrane.

• 8. Immunoblot the membrane with anti-calnexin antibody to determine which fractions contain ER.

• 9. Immunoblot the membrane with anti-GS28 antibody to determine which fractions contain cis-Golgi and to verify ER is not contaminated with cis-Golgi.

17.3.2.2.1 PCTV Budding Assay

• 1. Use a Bradford assay to determine the concentration of the ER and cytosol fractions (25).

• 2. Take 400–500 μg of ER protein (∼50 μL of a 10 mg/mL ER fraction) and combine with 40 μL of cytosol (40 μL of cytosol buffer for negative control), 100 μL of ATP mixture, 25 μL of 2.5 mM Mg²⁺, 25 μL of 2.5 mM Ca²⁺, 50 μL of 5 mM DTT, 10 μL E₆₀₀ prep, and 200 μL of transport buffer for a total volume of 500 μL.

• 3. Agitate slightly and incubate at 35–37°C for 35 min. Remove all air bubbles.

• 4. During incubation, prepare a continuous gradient of 0.1–1.15 M sucrose (10 mL total).

• 5. Stop the reaction by placing tubes on ice. Add 700 μL cold 10 mM HEPES.

• 6. Overlay suspension on gradient.

• 7. Centrifuge at 80,000 × g (Beckman SW41Ti rotor) for 95 min.

• 8. Remove the top 100 μL-containing cytosolic proteins and discard.

• 9. Fractionate by unloading 23 × 500 μL aliquots from the top of the tube.

• 10. Take 100 μL of each fraction and count the DPM for 2–3 min.

• 11. Plot DPM vs. fraction. Pool fractions with PCTVs.

17.3.3 Isoelectric Focusing

• 1. Add 2 volumes of lysis buffer to 500 μg of microsomal protein.

• 2. Lyse with 25-gauge syringe ten times.

• 3. Centrifuge at 15,000 × g for 10 min.

• 4. Take the supernatant. Add 1 volume of acetone.

• 5. Incubate at −20°C for 2 h.

• 6. Centrifuge at 15,000 × g for 10 min.

• 7. Discard the supernatant. Solubilize the pellet in 30 μL of BioRad Reagent 3, which can dissolve the most insoluble proteins. Shake 5 min.

• 8. Add 420 μL rehydration buffer and a pinch of bromophenol blue.

• 9. Place (+) acrylamide side of 24 cm Immobiline Drystrip gel face down in drystrip holder. Remove all air bubbles.

• 10. Cover with 5 mL Drystrip Cover Fluid.

• 11. Seal with Saran wrap and let sit for 12 h.

• 12. Place strip (+) side upwards, polyacrylamide side up onto IEF unit.

• 13. Attach metal brackets to contact acrylamide at each end of the strip.

• 14. Overlay with 4 mL of cover fluid.

• 15. Use the following Step and Hold pattern:

  • (a) Step and Hold 30 V for 1 h

  • (b) Step and Hold 150 V for 1 h

  • (c) Step and Hold 300 V for 1 h

  • (d) Step and Hold 1,000 V for 1 h

  • (e) Step and Hold 2,500 V for 1 h

  • (f) Gradient 8,000 V for 1 h

  • (f) Step and Hold 8,000 V for 3 h

  • (h) Step and Hold 150 V for 40 h

• 16. Incubate the IPG strip at room temperature in equilibration buffer with 1% DTT for 15 min. Wash 3 × 10 min with distilled water.

• 17. Incubate the IPG strip in equilibration buffer with 4.8% iodoacetamide for 15 min. Wash 3 × 10 min with distilled water.

17.3.4 -Dimensional Polyacrylamide Gel Electrophoresis

• 1. Use instructions in Ettan DALTsix electrophoresis unit to prepare the 8% SDS-PAGE gel. In short, mix distilled water, 1% APS, 1.5 M Tris–HCl, 40% acrylamide, and TEMED. Pour solution between glass plates and leave space for a stacking gel. Overlay with distilled water.

• 2. After 30 min, prepare the stacking gel by dissolving 0.5% agarose in 1× running buffer. Allow gel to cool to lukewarm temperature. Seal the IPG strip in agarose. Insert a single-well lane from a well comb for the prestained molecular weight marker. The stacking should polymerize within a few minutes.

• 3. Insert the gel(s) into the PAGE tank. Fill the upper and lower chambers with 1× running buffer.

• 4. Carefully remove the well lane. Load the lane with the molecular weight marker.

• 5. Complete the assembly of the gel unit and connect to a power supple. Run at 60 V until dye front reaches the bottom.

• 6. Following electrophoresis, stain the gel with Deep Purple Total protein Stain according to the manufacturer’s protocol (Amersham Biosciences).

• 7. Use a Typhoon 9400 imager to view the 2DE gels using wavelengths of 457 nm for excitation and 610 nm for emission.

17.3.5 Mass Spectrometry Sample Preparation

• 1. Cut a pipette tip to size with a scalpel. Use this to excise protein spots from the 2DE gel.

• 2. Eject the protein plug from the pipette tip into a 2-mL tube by inserting another pipette tip to pop it out. At this point the gel plug can be stored at −20°C if necessary.

• 3. Crush the gel plug in the bottom of the tube with a plunger from a small syringe.

• 4. Destain the plug with 5 × 15 min washes of 1 mL 30 mM ammonium bicarbonate/40% acetonitrile on an orbital shaker.

• 5. Dry the gel in a SpeedVac centrifuge until it turns to a powder (approximately 1.5 h).

• 6. Dissolve 2 μL of 0.2 μg/μL modified sequencing-grade trypsin (Promega) in 50 mM acetic acid, warmed first to 30°C for 15 min, and add to the dried gel.

• 7. Next, add 50 μL of 50 mM ammonium bicarbonate. Put the tube on ice for 1 h.

• 8. After the gel has taken up all the liquid, add an additional 25 μL of 50 mM ammonium bicarbonate/1 mM CaCl₂ solution. It is important to keep the gel pieces in excess liquid for the digestion process. Place the tube in a water bath set at 37°C for 18 h.

• 9. After the digestion, take the supernatant and set aside in a 1.5-mL tube.

• 10. Treat the remaining gel with 50 μL of the following solutions: 50 mM ammonium bicarbonate for one wash, 50% acetonitrile/1% trifluoroacetic acid for two washes, 20% formic acid/15% 2-propanol/25% acetonitrile for one wash, and 80% acetonitrile for one wash. After adding the solution for each wash, vortex the tube for 10 min, spin down, and then let it sit at room temperature for 15 min.

• 11. Extract the supernatant and pool with the first extract in the 1.5-mL tube. Dry the pooled extract in a SpeedVac for approximately 2 h. The samples can be stored at −20°C this point if necessary.

• 12. Add 20 μL of 0.8 M guanidine chloride/2.5% TFA to the dried sample and sit at room temperature for 15 min.

• 13. Sample clean-up using μZT should not be performed more than one day before MS analysis. Wet the μZT by two cycles of 50% acetonitrile solution and equilibrate with two cycles of 0.1% trifluoroacetic acid.

• 14. Take 5 μL of the solubilized sample and insert into the μZT for MALDI-TOF mass fingerprinting. The peptides in the sample will bind to the C₁₈ beads by fifteen cycles of the sample solution.

• 15. Wash the peptides bound to the beads by five cycles of 0.1% trifluoroacetic acid solution.

• 16. Elute the peptides from the column by fifteen cycles of 5 μL of 0.1% trifluoroacetic acid/50% acetonitrile solution. Do not introduce any air bubbles into the column.

• 17. Spot 0.3 μL of the sample on the MALDI target and allow to dry. Next, spot 0.3 μL of the CHCA matrix solution on top of the sample and allow to dry. Spot each sample in triplicate.

• 18. Analyse the extract by applying it to a Micromass MALDI-Q-TOF. The resulting peptide map can be searched against the Profound protein database. PROWL ProFound search engine available at http://prowl.rockefeller.edu/, using a focused rodent or mammalian search.

• 19. If proteins cannot be identified through database mining, tandem mass spectrometry (Chapter “Shotgun Protein Analysis by Liquid Chromatography-Tandem Mass Spectrometry”) can be used to sequence and identify the proteins. The resulting spectra can then be used to identify protein candidates in the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database with the MASCOT search engine (Matrix Science, London).

17.3.6 Liquid Chromatography Tandem Mass Spectrometry Sample Preparation

• 1. Dissolve 50 μg protein in 6 M urea/2 M thiourea solution with protease inhibitor cocktail (PI).

• 2. Lyse with 25-gauge syringe ten times.

• 3. Add 1 volume of DTT stock solution and incubate at room temperature for 30 min.

• 4. Add 1 volume of iodoacetamide stock solution and incubate at room temperature for 20 min.

• 5. Add 1 μg LysC and incubate for 3 h or overnight at room temperature.

• 6. Dilute sample with 4 volumes of digestion buffer. Add 1 μg trypsin and incubate overnight at room temperature. Digested peptides can be stored at −20°C.

• 7. Dry the sample in a SpeedVac.

• 8. Reconstitute the peptides in 150 μL mobile phase and 20 μL injected into the LC-MS/MS system. The mass spectrometer is equipped with an ion-spray source and operates in the positive ion mode.

• 9. The resulting spectra can be used to identify protein candidates in the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database with the Mascot search engine (Matrix Science, London).

17.4 Notes

• 1. Store Cell Recovery Solution, Buffers A-C, sucrose buffers, cytosol and transport buffers, and resolving gel buffer at 4°C.

• 2. For PCTV Budding Assay, store E₆₀₀ prep at −20°C and ATP mixture at −80°C. Also, store PCTVs at −80°C.

• 3. To prepare protease inhibitor cocktail (PI), dissolve one tablet in 2 mL of distilled water and use as 1:50 (PI cocktail: buffer).

• 4. To prepare 10% BSA-10 mM oleate complex:

  • (a) Dissolve 1 g BSA in 9 mL of 10 mM HEPES, pH 7.2.

  • (b) In a separate tube, dissolve sodium oleate in 1 mL of lukewarm distilled water.

  • (c) Add oleate to BSA mixture. Vortex.