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. Author manuscript; available in PMC: 2023 Oct 11.
Published in final edited form as: Methods Mol Biol. 2023;2603:219–234. doi: 10.1007/978-1-0716-2863-8_18

Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detection of Transient Protein Interactions

Jolene M Duda 1, Stefani N Thomas 2
PMCID: PMC10567058  NIHMSID: NIHMS1933681  PMID: 36370283

Abstract

Antibody-based affinity purification is a recognized method for use in studying protein–protein interactions. There are four different classes of proteins that are typically identified with such affinity purification workflows: bait protein, proteins that specifically interact with the bait protein, proteins nonspecifically associated with the antibody, and proteins that cross-react with the antibody. Mass spectrometry can be used to differentiate these classes of proteins in affinity-purified mixtures. Here we describe the use of stable isotope labeling by amino acids in cell culture, substrate trapping, and mass spectrometry to enable the objective identification of the components of affinity-purified protein complexes.

Keywords: SILAC, Substrate trapping, Mass spectrometry, Bait protein, Affinity purification, AAA+ ATPases, Protein, Protein interactions

1. Introduction

A well-known method to study protein–protein interactions includes antibody-based affinity purification of protein complexes, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and protein identification using mass spectrometry. The four different classes of proteins that are identified with such affinity purification workflows include bait protein, proteins that specifically interact with the bait protein, proteins that are nonspecifically associated with the antibody, and proteins that cross-react with the antibody [1]. Mass spectrometry (MS) can be used to identify the individual proteins in affinity-purified mixtures. The inclusion of stable isotope labeling enables the quantitation of proteins in these studies [2]. Stable isotope labeling by amino acids in cell culture (SILAC) provides various labeling conditions in which peptides can be quantified on a relative basis when comparing their corresponding MS1 peak intensities. Accordingly, the four different classes of proteins in affinity purification workflows, as mentioned earlier, can be distinguished based on their “heavy” to “light” peptide ratios [3].

Commonly used methods in affinity purification/mass-spectrometry protein interaction identification strategies include the use of an antibody immobilized by solid support such as resin, agarose, or magnetic beads or the use of an epitope tag [4, 5]. A considerable challenge with using these methods is the occurrence of antibody cross-reactivity resulting in background contaminants, or nonspecific binding proteins, which are co-purified and subsequently identified by mass spectrometry. The use of negative controls such as an siRNA-mediated depletion of the bait protein can help to filter some contaminants. However, it is not uncommon for the negative controls to fail [5, 6]. Tandem affinity purification (TAP) is a method that was developed to achieve improved differentiation between interacting proteins and contaminants, but it has limitations in identifying transient interactions [7, 8].

Substrate trapping is a strategy that allows for the stabilization of endogenous, low abundance, transiently-associated membrane protein complexes. This approach relies on the stringent filtering of quantitative mass spectrometry data to select high-quality protein interactor candidates. We previously conducted a study using a tetracycline-inducible HEK293 cell line that conditionally expressed a mutant form of an AAA+ ATPase with abrogated ATP hydrolysis function, Vps4B E235Q, rendering the protein unable to catalyze dissociation from its transiently interacting binding partners [3]. AAA+ ATPases make up a large family of proteins with diverse functionality. One commonality among these proteins is the function of ATP hydrolysis in interacting with binding partners [9]. When this mutant AAA+ ATPase binds tightly to its substrate, the substrate is then “trapped” [10–12]. In our study, a Myc-tagged version of Vps4B E235Q was used as the bait protein, which permitted the detection of transient interacting proteins that would not otherwise be observable with traditional affinity purification strategies [13–17].

A workflow for performing protein quantification and characterization based on the ratios of SILAC-labeled peptides is presented in Fig. 1a. This figure focuses on the first three classes of proteins identified in affinity purification workflows as mentioned earlier: bait protein, proteins that specifically interact with the bait protein, and proteins that are nonspecifically associated with the antibody. The peptide ratios seen in Fig. 1b are relative abundance ratios based on time points of 0, 4, and 9 h of tetracycline induction of a bait protein, Vps4B E235Q. These different abundance ratios were used to characterize the three classes of proteins described earlier that are time-dependent on tetracycline induction. The relative abundance of the bait protein should increase with each time point. The proteins interacting with the bait protein should increase between the 0 and 4 h time point; however, between the 4 h and 9 h time points, there would be no further increase because of bait protein binding site saturation. Finally, the relative abundance ratios of the nonspecific binding proteins should remain the same between all of the time points because this mode of binding is not dependent on the tetracycline induction. Figure 2 illustrates the peptide ratios showing a distinct distribution of peptides from the bait protein, bait-interacting proteins, and the nonspecific binding proteins.

Fig. 1.

Fig. 1

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Workflow for SILAC-based substrate trapping. (a) Tetracycline-inducible HEK293 Vps4B (E235Q)-myc cells were cultured in light, medium, and heavy SILAC media, and they were induced with tetracycline for 0, 4, or 9 h, respectively. After the induction, cells from each condition were harvested, lysed, and combined, followed by immunoprecipitation and separation via SDS-PAGE. This was followed by in-gel digestion, LC-MS/MS, and relative protein quantification. (b) The bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins were identified and distinguished based on their relative peptide abundance ratios. (Reproduced from Ref. [3] with permission of The American Chemical Society)

Fig. 2.

Fig. 2

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Peptide ratios have separate distributions based on the class of protein. (a) The bait protein peptides are the only peptides distributed with a ratio > 2.0 in the 9 h/4 h time set. This indicates a time-dependent increase in Vps4B. The ratios of the bait-interacting proteins were approximately 1.0, indicating saturation of binding. The relative abundances of the bait protein and peptides belonging to the endogenous bait interacting proteins were >2.0 at the (b) 4 h/0 h and (c) 9 h/0 h induction times, which was reflective of the time-dependent increase in abundance from 0 h; however, at all three relative time points the nonspecific binding protein ratio remained at 1.0. (Reproduced from Ref. [3] with permission of The American Chemical Society)

Although the previously conducted study focused on Vps4B, the following approach is applicable to other protein–protein interaction systems in which protein binding is related to ATP hydrolysis activity. The approach described combines SILAC with tetracycline-inducible bait protein expression, immunoprecipitation, and gel electrophoresis, followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS)-based quantification to enable the objective identification of the components of purified protein complexes.

2. Materials

2.1. Cell Culture

  1. Supplemented SILAC cell culture medium: one 500 mL arginine- and lysine-deficient bottle of SILAC Dulbecco’s modified Eagle’s medium (DMEM) will be needed for each condition. Remove the necessary volume of SILAC DMEM for each amino acid to prepare a stock concentration of 100 μg/mL and add directly to the amino acid vial to dissolve each amino acid used. The amino acids to be added for each condition are listed in Subheading 2.1, items 2–4. Remove 55 mL of the SILAC DMEM and replace it with 50 mL dialyzed fetal bovine serum (FBS) and 5 mL penicillin/streptomycin. Add the dissolved amino acids, 5 μg/mL blasticidin, and 125 μg/mL zeocin to the SILAC DMEM and mix well. Filter through a 0.22 μm flask into a newly labeled bottle. Store supplemented SILAC medium at 4 °C for 3–6 months.

  2. Unlabeled “light”-condition cells: supplemented SILAC medium with 12C6-L-lysine • 2HCl (Lys0;100 μg/mL) and 12C6-L-arginine • HCl (Arg0;100 μg/mL).

  3. Labeled “medium”-condition cells: supplemented SILAC medium with D4-L-lysine • 2HCl (Lys4; 100 μg/mL) and 13C6-L-arginine • HCl (Arg6; 100 μg/mL).

  4. Labeled “Heavy”- condition cells: supplemented SILAC medium containing 13C6, 15N2-L-lysine • 2HCl (Lys8; 100 μg/mL) and 13C6, 15N4 L-arginine • HCl (Arg10; 100 μg/mL).

  5. Phosphate-buffered saline (PBS).

  6. 0.05% Trypsin/EDTA.

  7. T-REx™ System [15, 18].

  8. Tetracycline: 0.75 μg/mL.

2.2. Cell Lysis

  1. Kimble pellet pestle motor.

  2. Immunoprecipitation (IP) Buffer: 20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 5 mM ATP, 2 mM MgCl2, protease inhibitor cocktail EDTA-free (1 mini tablet per 10 mL).

  3. Phosphate-buffered Saline (PBS).

2.3. Immunoprecipitation (IP)

  1. IP buffer: see Subheading 2.2, item 2.

  2. IP wash buffer: IP buffer with 1% TritonX-100.

  3. 1× Tris-buffered saline (TBS).

  4. Profound™ Mammalian c-Myc Tag IP kit and application set.

  5. Anti-c-myc agarose slurry: 1:100 (w/w) anti-c-Myc/protein.

  6. Protein A agarose: 50 μL of agarose slurry/1 mg protein.

  7. 100 mM NH4HCO3: dissolve 0.158 g in 20 mL of HPLC-grade water (this same solution can be used again for Subheading 2.6).

  8. 1 M Dithiothreitol (DTT): dissolve 7.713 mg of DTT in 50 μL of HPLC-grade water.

  9. 2× Nonreducing sample buffer: prepared from 5× Immuno-Pure Lane marker nonreducing sample buffer included with IP kit.

2.4. Total Protein Quantitation, SDS-PAGE, and In-Gel Digestion [19]

  1. BCA Protein Assay Kit.
    1. Working reagent: 50 parts reagent A to 1 part reagent B.
    2. BSA stock solution: 2000 μg/mL.
    3. BCA standard curve: using the BSA stock solution prepare a 7 points curve with serial dilutions: 2000 μg/mL (stock), 1500 μg/mL, 1000 μg/mL, 750 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL, 0 μg/mL (blank).

  2. Precast Bolt 4–12% Bis-Tris gels.

  3. SimplyBlue SafeStain Coomassie G-250 stain.

  4. Bolt MES running buffer.

  5. SeeBlue Plus2 prestained standard.

  6. Bolt LDS sample buffer.

  7. Gel-loading pipette tips.

  8. Digestion solution: Add 50 μL water to a 1.5 mL microcentrifuge tube, then add 50 μL of 100 mM NH4HCO3, 5 μL of CaCl2, and 1.5 μg of trypsin. Mix by vortexing.

  9. Digestion buffer: digestion solution without trypsin.

  10. 100 mM NH4HCO3: see Subheading 2.3.

  11. 50 mM NH4HCO3: Mix 500 μL of HPLC-grade water with 500 μL of 100 mM NH4HCO3.

  12. 10 mM Dithiothreitol: dissolve 1.5 mg in 1 mL of 100 mM NH4HCO3 (prepare immediately before use).

  13. 55 mM iodoacetamide: dissolve 10 mg in 1 mL of 100 mM NH4HCO3 (prepare immediately before use, light sensitive).

  14. 5% Formic acid: add 950 μL of water to a labeled 1.5 mL microcentrifuge tube, then add 50 μL of formic acid to the tube. Mix by vortexing.

  15. Acetonitrile.

2.5. Desalting Using C18 Stop and Go Extraction (STAGE) Tips [20–22]

  1. Solvent A: 0.1% formic acid: add 9.8 mL of HPLC-grade water to a 10 mL glass vial, then add 200 μL of formic acid. Mix by inversion.

  2. Solvent B: 50% acetonitrile/0.1% formic acid: add 499 μL of HPLC-grade water to a 10 mL glass vial, then add 500 μL of acetonitrile and 1 μL of formic acid. Mix by inversion.

  3. Solvent C: 3% acetonitrile/0.1% formic acid: in a 10 mL glass vial add 969 μL of HPLC-grade water, then add 30 μL of acetonitrile and 1 μL of formic acid. Mix by inversion.

2.6. Immunoblot Analysis

  1. SDS PAGE: (see Subheading 2.4).

  2. Nitrocellulose membranes.

  3. Blocking buffer: PBS/0.1% Tween-20/5% nonfat milk (see Note 1).

  4. Washing buffer: PBS or TBS containing 0.1% Tween-20.

  5. Chemiluminescent detection: SuperSignal West Pico chemiluminescent substrate.

2.7. Antibodies (See Note 2)

  1. Primary antibodies: Vps4B, ESCRT-III complex protein antibodies, c-myc, and actin.

  2. Secondary antibodies, HRP-conjugated.

2.8. LC-MS/MS [23]

  1. Formic acid.

  2. LC-MS-grade acetonitrile.

  3. LC-MS-grade water.

  4. Solvent A: 3% acetonitrile/0.1% formic acid in water.

  5. Solvent B: 90% acetonitrile/0.1% formic acid.

  6. C18 analytical column: 50 cm length × 75 μm i.d. 2 μm Acclaim PepMap RSLC C18 EASY-Spray column.

  7. Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) (see Note 3).

  8. Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer with an EASY-Spray nanospray ionization source (Thermo Fisher Scientific) (see Note 4).

2.9. Database Searching/Protein Identification [24]

  1. MaxQuant software (www.maxquant.org) (see Note 5).

3. Methods

3.1. Establishing Tetracycline-Inducible Cell Line

  1. Tetracycline-inducible HEK293 stable cell lines expressing c-myc-tagged Vps4B(E235Q) were generated as described previously [15] using the T-REx tetracycline-regulated expression system [18].

  2. Vps4B expression in Escherichia coli: insert mouse Vps4B cDNA into plasmid pHO4d between NcoI and EcoRI sites with amino acids PNSG between the C-terminus of Vps4B and the His6-Myc tag.

  3. Generate E235Q mutation using QuikChange ™ site-directed mutagenesis (Stratagene).

  4. To express Vps4B WTor E235Q in mammalian cells, insert the corresponding cDNA into pEGFP-N1 (Takara Bio) between XhoI and BamHI sites.

  5. To create tetracycline-regulated constructs, the entire Vps4B-cmyc fusion is amplified and inserted into pcDNA4/TO using BamHI and XhoI sites.

  6. Generate untagged constructs by cloning into pcDNA3.1(+) between BamHI and XhoI sites.

  7. Generate Myc-tagged constructs by cloning into pcDNA3.1/Myc-His(−)A between XbaI and HindIII sites with a linker of KLGP between hSnf7–1/hVps4B and the Myc epitope.

  8. Maintain Vps4B(E235Q)-c-Myc cells in supplemented SILAC DMEM media.

3.2. SILAC-Based Cell Culture

  1. Culture “light”-condition cells in supplemented SILAC medium containing 12C6-L-lysine • 2HCl (Lys0; 100 μg/mL) and 12C6-L-arginine • HCl (Arg0; 100 μg/mL) for at least five cell doublings in 10 × 150 mm culture dishes.

  2. Culture “medium”-condition cells in supplemented SILAC medium containing D4-L-lysine • 2HCl (Lys4; 100 μg/mL) and 13C6-L-arginine • HCl (Arg6; 100 μg/ mL) for at least five cell doublings in 10 × 150 mm culture dishes.

  3. Culture “heavy”- condition cells in supplemented SILAC medium containing 13C6, 15N2-L-lysine • 2HCl (100 μg/mL) (Lys8) and 13C6, 15N4 L-arginine • HCl (Arg10; 100 μg/mL) for at least five cell doublings in 10 × 150 mm culture dishes.

  4. Remove media from the dish by aspirating.

  5. Rinse cells with 5 mL of PBS to ensure complete media removal.

  6. Add 3 mL of 0.25% trypsin/EDTA to the culture dish, and incubate at 37 °C/5% CO2 until cells are detached from the surface (~2–5 min).

  7. After cells are detached, add 23 mL of appropriate media for the SILAC condition and transfer to a 50 mL conical tube.

  8. Centrifuge the conical tube for 5 min at 1500 rpm, so the cells form a pellet.

  9. Discard the supernatant, and dissolve the pellet in 10 mL of fresh medium.

  10. Add 24 mL of medium to a newly labeled plate.

  11. Transfer 2 mL of the cells from the conical tube to the plate and return to the incubator.

  12. Repeat for each plate/condition.

  13. Maintain the cells at 37 °C and 5% CO2 in a humidified incubator.

  14. Allow cells to grow for five doublings. The incorporation of the isotope should be >95%.

  15. Harvest and lyse a portion of the cells (1 × 106), keeping the various labeled conditions separate (see Subheading 3.3).

  16. Perform a BCA protein assay to quantitate the protein (see Subheading 3.4).

  17. Load the samples into separate wells in an SDS PAGE gel and perform in-gel digestion of the protein sample (see Subheading 3.6 and Note 6).

  18. Determine incorporation of stable isotope-labeled amino acids by LC-MS/MS analysis of tryptic peptides isolated from the labeled cells (see Subheading 3.9).

  19. After stable-isotope incorporation has been verified, induce cells to obtain Vps4B (E235Q) expression for 4 h (SILAC “medium” cells) or 9 h (SILAC “heavy” cells) by incubating cells with 0.75 μg/mL tetracycline. Cells cultured in SILAC “light” medium should not be induced (0 h, control).

3.3. Cell Lysis

  1. Remove media from the first dish by tilting the dish and aspirate or remove with a 25 mL pipette. Discard media.

  2. Rinse the dish with 5 mL of cold PBS.

  3. Add 1 mL of IP buffer and detach cells from the dish using a cell scraper.

  4. Transfer the cell suspension into a labeled tube and lyse the cells using a motorized pestle homogenizer.

  5. Repeat for each label condition.

  6. Pellet insoluble material by centrifugation at 800 × g for 5 min at 4 °C.

  7. Carefully decant the supernatant into a newly labeled conical tube and save the pelleted material.

  8. Use supernatant aliquot for BCA protein assay.

3.4. Determination of Protein Concentration by BCA Protein Assay

  1. Add 20 μL of HPLC-grade water to a 1.5 mL microcentrifuge tube, then add 5 μL of sample to create a 1:5 dilution.

  2. Vortex sample and briefly centrifuge to recollect sample at the base of the tube.

  3. Add 10 μL of sample to a 96-well flat-bottom plate in duplicate.

  4. Plate 10 μL each of a 7-point BSA standard curve, ranging from 2000 μg/mL to 125 μg/mL, plus 0 μg/mL (blank) in duplicate.

  5. Prepare BCA reagent by mixing 50 parts BCA reagent A and 1 part BCA reagent B (see Note 7).

  6. Add 200 μL of the BCA reagent to make a 1:20 working reagent ratio (v/v) (see Note 8).

  7. Incubate the 96-well plate at 37 °C for 30 min.

  8. After 30 min, scan the plate using a plate reader at 562 nm to determine the protein concentrations of the samples.

  9. Once protein concentration is determined, dilute the sample with IP buffer to reach a concentration of 8 μg/μL.

3.5. Immunoprecipitation (IP)

  1. Combine an equivalent amount of protein from the SILAC light, medium, and heavy cell lysates into a 1.5 mL microcentrifuge tube and preclear overnight with rotation at 4 °C with a protein A agarose. A total of >500 μg of protein is required (see Note 9).

  2. Prepare a positive control by adding 50 μL c-Myc-tagged positive control from the IP kit and diluting it in 150 μL TBS.

  3. Place bottom plugs on Handee™ Mini-Spin Columns from IP kit.

  4. Add cell lysate sample and positive control to the spin columns.

  5. Invert the vial containing the anti-c-Myc agarose beads several times to resuspend the beads prior to dispensing.

  6. Add 10 μL anti-c-Myc agarose slurry into the spin column with the positive control (see Note 10).

  7. Incubate the samples overnight with rotation (end-over-end) at 4 °C.

  8. Loosen the cap on the spin column and remove the bottom plug.

  9. Place the spin column inside of a collection tube and centrifuge for 10 sec. If desired, collect and save supernatant.

  10. Add 0.5 mL IP wash buffer with 1% Triton X-100. Place a bottom plug on the spin column.

  11. Loosely screw on the cap of the spin column and gently invert three times.

  12. Place spin column in the new collection tube. Remove the bottom plug. Pulse centrifuge for 10 s. Discard flow-through.

  13. Repeat steps 9–12 twice.

  14. Add 25 μL 2× nonreducing buffer prepared from 5× Immunopure lane marker nonreducing sample buffer from IP kit. Place a bottom plug on the spin column (see Note 11).

  15. Transfer spin column to new collection tube.

  16. Incubate at 95 °C for 5 min.

  17. Remove the bottom plug. Pulse centrifuge for 10 s to elute the immunoprecipitated proteins.

  18. Set aside an aliquot of the immunoprecipitated proteins for use in Subheading 3.8.

3.6. In-Gel Digestion [19]

  1. Separate the proteins by SDS PAGE and stain gel with Coomassie Blue.

  2. Excise bands of interest from SDS-PAGE gel using a plastic gel cutting razor blade (see Note 12).

  3. Cut gel bands into 1 × 1 mm pieces and place them into a 1.5 mL methanol-rinsed microcentrifuge tube.

  4. Wash gel pieces with 75 μL 100 mM NH4HCO3 for 15 min with shaking.

  5. Shrink gel pieces by adding 75 μL acetonitrile.

  6. Dry gel pieces in a vacuum centrifuge for 15 min.

  7. Reduce disulfide bonds with 75 μL 10 mM DTT for 1 h at 60 °C.

  8. Alkylate cysteines with 75 μL 55 mM iodoacetamide for 45 min at room temperature in the dark.

  9. Wash gel pieces with 75 μL 100 mM NH4HCO3.

  10. Shrink with 75 μL acetonitrile.

  11. Dry gel pieces in a vacuum centrifuge for 15 min.

  12. Rehydrate gel pieces with enough digestion solution to cover gel pieces on ice for 45 min.

  13. Remove remaining supernatant.

  14. Add 25 μL of digestion buffer and let incubate overnight at 37 °C.

  15. Centrifuge gel pieces and transfer supernatant to another 1.5 mL methanol-rinsed microcentrifuge tube. Add supernatant from each extraction in Step 15 to this tube.

  16. Extract peptides at 37 °C for 15 min with shaking at 400 RPM: once with 70 μL 50 mM NH4HCO3, pH 7.8, and twice with 5% formic acid/acetonitrile.

3.7. Desalting Extracted Peptides [20–22]

  1. Prepare C18 STAGE tips using a 16-gauge blunt end needle to hole-punch two ~1 mm disks of C18 material. Use the puncher to pack the two disks of C18 material into the tip of each STAGE tip (200 μL pipette tip) for a total binding capacity of 20 μg. Use adaptors to hold the STAGE tip at the opening of each 2 mL microcentrifuge tube.

  2. Condition each STAGE tip by adding 100 μL of acetonitrile.

  3. Centrifuge for 3 min at 3000 × g at room temperature, then discard the solution from the collection vial (see Notes 13 and 14).

  4. Wash the STAGE tip with 100 μL of solvent B. Centrifuge for 3 min at 3000 × g at room temperature. Discard the flow-through.

  5. Equilibrate the STAGE tip twice with 100 μL of solvent A. Centrifuge for 3 min at 3000 × g at room temperature after each solvent loading. Discard the flow-through.

  6. Reconstitute the sample in 100 μL of solvent C, vortex briefly, and check to ensure the sample is completely dissolved.

  7. Add the sample to the STAGE tip and centrifuge at 3000 × g for 3 min at room temperature. Discard the flow-through.

  8. Wash the sample twice with 100 μL of solvent A. Centrifuge at 3000 × g for 3 min at room temperature. Discard the flow-through.

  9. Replace the collection vial with a new 1.5 mL methanol-rinsed microcentrifuge tube to collect the eluate.

  10. Elute the sample with 60 μL of STAGE tip solvent B. Centrifuge for 3 min at 3000 × g at room temperature.

  11. Dry the eluate completely in a SpeedVac.

  12. Once completely dry, store desalted peptides at −20 °C prior to LC-MS/MS analysis.

3.8. Immunoblot Analysis

  1. Separate IP eluate by SDS-PAGE.

  2. Transfer protein to nitrocellulose membranes.

  3. Block nonspecific binding sites on membranes for 1 h at room temperature in 10 mL of blocking buffer.

  4. Probe with primary antibody overnight at 4 °C.

  5. Wash 3 × 5 min with wash buffer.

  6. Incubate with HRP-conjugated secondary antibody for 1 h at room temperature.

  7. Wash 3 × 5 min with wash buffer.

  8. Follow manufacturer’s recommendations for preparing and adding chemiluminescent detection reagent.

3.9. LC-MS/MS [23]

  1. Utilize an LC method with the following parameters:
    1. Gradient pump flow rate = 0.3 μL/min; loading pump flow rate = 5.0 μL/min.
    2. Column heater temperature = 35 °C.
    3. Autosampler temperature = 6.0 °C.
    4. Separation gradient: 60 min linear gradient from 3 to 35% solvent B, followed by 35–90% solvent B in 5 min, 90% solvent B for 5 min, and column re-equilibration with 3% solvent B for 10 min.
  2. Create an MS data acquisition method with the following parameters:
    1. Full MS
      1. Polarity: positive.
      2. Orbitrap resolution: 60,000 at 200 m/z.
      3. Automatic gain control (AGC) target: 4 × 105 ion counts.
      4. Maximum injection time: 50 ms.
      5. Scan range: 350–1800 m/z.
    2. Data-dependent MS/MS
      1. Detector type: Orbitrap.
      2. Resolution: 7500.
      3. Activation type: HCD.
      4. AGC target: 5 × 104 ion counts.
      5. Maximum injection time: 50 ms.
      6. Number of dependent scans: 10.
      7. Isolation window: 0.7 m/z.
      8. Dynamic exclusion: 45 s.
      9. Charge exclusion: Include 2–6 charge states.

3.10. SILAC-Labeled Protein Identification and Quantitation

  1. Conduct database search using MaxQuant [24] or another database search engine using the following parameters:
    1. Database: UniProtKB Homo sapiens protein database with reviewed sequence entries.
    2. Enzyme cleavage specificity: trypsin.
    3. Fixed modification: Cys +57.02146 (carbamidomethylation).
    4. Variable modifications: Lys +4.02511 (D4), Lys +8.01420 (13C6,15N2), Arg +6.02013 (13C6), Arg +10.00827 (13C6,15N4), Met +15.99491 (oxidation), Protein N-terminus +42.01056 (acetylation).
    5. Precursor mass tolerance: 20 ppm.
    6. Product mass tolerance: 20 ppm.
    7. Maximum missed cleavages: 2.
    8. Maximum charge: 5.
    9. Peptide FDR: 1%.

  2. Select option for peptide and protein quantification based on the SILAC labels used in the experiment [24].

  3. Researchers are encouraged to deposit their raw mass spectrometry data to publicly-accessible repositories such as the ProteomeXchange Consortium via the PRIDE partner repository [25].

Acknowledgments

SNT acknowledges funding from the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and start-up funds from the University of Minnesota Department of Laboratory Medicine and Pathology.

4 Notes

1.

Some antibodies require dilution in 5% BSA instead of 5% milk for Western blotting.

2.

These antibodies are specific for the analysis of Vps4B E235Q binding proteins. Investigators should select antibodies that are known interacting proteins of their bait protein of interest.

3.

Other LC systems that can deliver nanoflow rates and can operate up to a pressure of 1000 bar can be used for peptide separation.

4.

Other mass spectrometry systems can be used as well.

5.

Other software packages can be used that support the identification and SILAC-based quantification of peptides and proteins from high-resolution LC-MS/MS data.

6.

In-solution digestion may be performed as well but will not be discussed in this protocol.

7.

This solution must be made immediately prior to use.

8.

1:20 ratio is important only if there are interfering substances present in the sample/buffer. The ratio can be either 1:20 (200 μL working reagent) or 1:8 (80 μL working reagent). Although, if there are inhibiting substances present in the samples, such as chelators, a 1:20 dilution may still not be enough of a dilution, and a further dilution should be utilized.

9.

Preclearing is useful in order to remove contaminating noninteracting proteins; however, the choice to preclear depends on the method of affinity purification used [4].

10.

Use 20 μL of anti-c Myc slurry for every 1 mg of protein.

11.

After completing 3.5.14, add 3 μL of 1 M DTT to the sample for SDS-PAGE analysis under reducing conditions.

12.

The particular method of gel band excision will depend on the proteins being separated by gel electrophoresis and the density of the gel bands.

13.

If necessary, decrease the centrifugation speed to prevent the STAGE tip extraction disks from drying out.

14.

If no liquid passes through the STAGE tip following centrifugation, this indicates that the STAGE tip is packed too tightly. Discard the STAGE tip and use a new one.

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