Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

1. Immunoprecipitation

NOTE: We used non-sodium dodecyl sulfate (SDS) lysis buffer and citrate elution, as described in the following sections. However, the use of an alternative in-house immunoprecipitation technique may be also applicable for preparing LC-MS/MS samples.

1. Transfect cultured cells with vectors encoding an epitope tag alone or a fusion protein. For acquiring representative data, transfer J774 cells (1 x 10⁶) with vectors encoding green fluorescent protein (GFP) alone or GFP-fused Capn6 (calpain-6 gene) using cationic liposomes.
2. Conjugate antibody to magnetic beads.
   1. For this purpose, mix anti-GFP antibody (2 μL/reaction) with magnetic beads (25 μL/reaction) in 500 µL of citrate phosphate buffer (24.5 mM citric acid, 51.7 mM dibasic sodium phosphate; pH 5.0) in a 1.5 mL test tube.
   2. Rotate the mixture at 50 rpm for 1 h at room temperature. Then, wash the IgG-conjugated beads three times with citrate phosphate buffer containing 0.1% Polyoxyethylene (20) sorbitan monolaurate.
3. Lyse the transfected cells using an appropriate lysis buffer. For acquiring representative data, lyse the transfected cells for 30 min on ice in 400 µL of lysis buffer (50 mM Tris-HCl; pH 7.5, 120 mM NaCl, 0.5% poly(oxyethelene) octylphenyl ether) containing 40 μmol/L phenylmethylsulfonyl fluoride, 50 μg/mL leupeptin, 50 μg/mL aprotinin, 200 μmol/L sodium orthovanadate, and 1 mM ethylene glycol tetraacetic acid (EGTA) in 1.5 mL test tubes 24 h after transfection.
4. Clear the lysate by centrifuging at 15,000 x g for 5 min and collect the supernatant.
5. Preclear the lysate. Add unlabeled magnetic beads (25 μL/reaction) in a 1.5 mL-test tube. Wash the beads three times with 500 µL of citrate phosphate buffer. After the removal of citrate phosphate buffer in final wash, add the cell lysate over the magnetic beads. Rotate the mixture using rotator at 50 rpm for 30 min at room temperature.
6. Place the test tube on a magnetic stand for 5 min for magnetic separation, and collect the cell lysate. Use of the magnetic stand designated by the manufacturer is recommended.
7. Bind the target proteins to the conjugated beads. Place the immunoglobulin (Ig)G-conjugated beads on a magnetic stand for 5 min for magnetic separation, discard the citrate buffer, and then add the cell lysate to the separated beads.
8. Rotate the mixture at 50 rpm for 1 h at room temperature.
9. Separate the target proteins from free non-target proteins. Wash the beads three times using 500 µL of citrate phosphate buffer containing 0.1% Polyoxyethylene (20) sorbitan monolaurate. After the final wash, add 30 µL of citrate buffer (pH 2–3), and incubate for 5 min at room temperature to elute the target proteins.
   NOTE: If recovery from the immunoprecipitation is insufficient, the use of alternative epitope tags, such as FLAG or HIS, may improve the efficiency. If the efficiency of the elution is insufficient, the use of other eluants, such as an SDS-based solution, may improve the efficiency.

2. On-membrane digestion of proteins

NOTE: Using protein-free materials and equipment is necessary to avoid contamination with exogenous proteins. In addition, it is recommended that the operator wear a surgical mask and gloves to avoid contamination by human proteins.

1. Cut PVDF membranes into 3 mm x 3 mm pieces using surgical scissors that have been wiped with methanol and dried immediately prior to use.
2. Add 2–5 µL of ethanol on the pieces of PVDF membrane on clean aluminum foil.
3. Before they dry completely, add the eluant (2–5 µL each, 4–6 pieces per sample) onto the hydrophilic PVDF membranes, and then air-dry the membranes until the membrane surface becomes matte. Dried membranes can be stored at 4 °C.
4. Transfer the all membranes into 1.5 mL plastic tubes, add 20–30 µL of ethanol to make the membranes hydrophilic, and then remove the ethanol using a pipette.
5. Before the membrane dries out completely, add 200 µL of dithiothreitol (DTT)-based reaction solution (80 mM NH₄HCO₃, 10 mM DTT, and 20% acetonitrile) and incubate it at 56 °C for 1 h.
6. Replace the reaction solution with 300 µL of iodoacetamide solution (80 mM NH₄HCO₃, 55 mM iodoacetamide, and 20% acetonitrile), and incubate for 45 min at room temperature in the dark.
7. Wash the membranes twice with 1 mL of distilled water and once with 1 mL of 2% acetonitrile by vortex mixing for >10 s.
8. Dissolve lyophilized MS-grade trypsin (Table of Materials) directly in the reaction solution (30 mM NH₄HCO₃ containing 70% acetonitrile). Add 100 µL of the reaction solution containing 1 μg of trypsin (Table of Materials) (30 mM NH₄HCO₃ containing 70% acetonitrile) and incubate at 37 °C overnight.
9. Transfer the reaction solution containing the tryptic digests into a clean 1.5 mL test tube using a pipette.
10. Add 100 µL of wash solution (70% acetonitrile/1% trifluoroacetic acid) to the membrane and incubate it at 60 °C for 2 h. Collect the wash solution and mix it with the reaction solution. Add another 100 μL of wash solution to the membrane and sonicate it for 10 min. Then collect the wash solution and mix with the reaction solution.
11. Cover the test tube with a piece of laboratory film and make a couple of small holes with a needle. Dry the solution using a vacuum concentrator.
12. Dissolve the residue in 10 µL of 0.2% formic acid. After centrifugation (12,000 × g, 3 min at room temperature), transfer the supernatant into a sample tube.
    NOTE: The washes are the critical steps of this section. During on-membrane protein digestion, the washing of the membranes containing the immobilized proteins after reduction and alkylation with distilled water, followed by 2% acetonitrile, for more than 10 sec each, is critical for the removal of the reagents.

3. LC–electrospray ionization (ESI)-MS/MS analysis

1. Activate an ESI-MS/MS instrument (Table of Materials) coupled with a nano-LC HPLC system (Table of Materials). Link a pre-column (Table of Materials) and an analytical column (Table of Materials).
2. Prior to the analysis, calibrate the mass spectrometer using tryptic digests of bovine serum albumin dissolved in 0.2% formic acid, which provides standard peptides.
3. Analyze the samples using positive ion mode and a mass range of 400–1,250 m/z; then acquire up to 10 MS/MS spectra (100 ms each) with a mass range of 100–1,600 m/z and a linear gradient of 2%–80% acetonitrile and 0.2% formic acid for 80 min at a flow rate of 300 nL/min.
4. Analyze the output data using software for protein identification (Table of Materials) to identify the candidate associated proteins. For the positive identification of a candidate protein, detection of at least one high-fidelity peptide fragment (> 95% fidelity) is required.
5. Omit the proteins that are similarly precipitated in GFP-expressing lysates (transfection of vector expressing a GFP tag alone) from the list of candidate proteins.
   NOTE: If few proteins are identified in the database analysis, modification of the MS/MS acquisition conditions (e.g., changing the time from 100 ms each to 50 ms each) may increase the number of proteins identified.