Name: Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique (Video) | JoVE Journal
Uploaded: 2019-07-19T13:30:00.000+00:00
Duration: 7 min 7 s
Description: Learn on-membrane digestion for proteomic analysis of protein-protein interactions using mass spectrometry. This method eliminates gel electrophoresis, improving throughput for crude immunoprecipitants.

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5 minutes

This study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant Number 17K09869 (to AM), Japan Society for the Promotion of Science KAKENHI Grant Number 15K09418 (to TM), a research grant from Kanehara Ichiro Medical Science Foundation and a research grant from Suzuken Memorial Foundation (all to TM).

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.
<ol>
	<li>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.</li>
	<li>Conjugate antibody to magnetic beads.
	<ol>
		<li>For this purpose, mix anti-GFP antibody (2 &#956;L/reaction) with magnetic beads (25 &#956;L/reaction) in 500 &#181;L 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Lyse the transfected cells using an appropriate lysis buffer. For acquiring representative data, lyse the transfected cells for 30 min on ice in 400 &#181;L of lysis buffer (50 mM Tris-HCl; pH 7.5, 120 mM NaCl, 0.5% poly(oxyethelene) octylphenyl ether) containing 40 &#956;mol/L phenylmethylsulfonyl fluoride, 50 &#956;g/mL leupeptin, 50 &#956;g/mL aprotinin, 200 &#956;mol/L sodium orthovanadate, and 1 mM ethylene glycol tetraacetic acid (EGTA) in 1.5 mL test tubes 24 h after transfection.</li>
	<li>Clear the lysate by centrifuging at 15,000 x g for 5 min and collect the supernatant.</li>
	<li>Preclear the lysate. Add unlabeled magnetic beads (25 &#956;L/reaction) in a 1.5 mL-test tube. Wash the beads three times with 500 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Rotate the mixture at 50 rpm for 1 h at room temperature.</li>
	<li>Separate the target proteins from free non-target proteins. Wash the beads three times using 500 &#181;L of citrate phosphate buffer containing 0.1% Polyoxyethylene (20) sorbitan monolaurate. After the final wash, add 30 &#181;L of citrate buffer (pH 2&#8211;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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Add 2&#8211;5 &#181;L of ethanol on the pieces of PVDF membrane on clean aluminum foil.</li>
	<li>Before they dry completely, add the eluant (2&#8211;5 &#181;L each, 4&#8211;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 &#176;C.</li>
	<li>Transfer the all membranes into 1.5 mL plastic tubes, add 20&#8211;30 &#181;L of ethanol to make the membranes hydrophilic, and then remove the ethanol using a pipette.</li>
	<li>Before the membrane dries out completely, add 200 &#181;L of dithiothreitol (DTT)-based reaction solution (80 mM NH4HCO3, 10 mM DTT, and 20% acetonitrile) and incubate it at 56 &#176;C for 1 h.</li>
	<li>Replace the reaction solution with 300 &#181;L of iodoacetamide solution (80 mM NH4HCO3, 55 mM iodoacetamide, and 20% acetonitrile), and incubate for 45 min at room temperature in the dark.</li>
	<li>Wash the membranes twice with 1 mL of distilled water and once with 1 mL of 2% acetonitrile by vortex mixing for &#62;10 s.</li>
	<li>Dissolve lyophilized MS-grade trypsin (Table of Materials) directly in the reaction solution (30 mM NH4HCO3 containing 70% acetonitrile). Add 100 &#181;L of the reaction solution containing 1 &#956;g of trypsin (Table of Materials) (30 mM NH4HCO3 containing 70% acetonitrile) and incubate at 37 &#176;C overnight.</li>
	<li>Transfer the reaction solution containing the tryptic digests into a clean 1.5 mL test tube using a pipette.</li>
	<li>Add 100 &#181;L of wash solution (70% acetonitrile/1% trifluoroacetic acid) to the membrane and incubate it at 60 &#176;C for 2 h. Collect the wash solution and mix it with the reaction solution. Add another 100 &#956;L of wash solution to the membrane and sonicate it for 10 min. Then collect the wash solution and mix with the reaction solution.</li>
	<li>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.</li>
	<li>Dissolve the residue in 10 &#181;L of 0.2% formic acid. After centrifugation (12,000 &#215; 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.</li>
</ol>
3. LC&#8211;electrospray ionization (ESI)-MS/MS analysis
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Analyze the samples using positive ion mode and a mass range of 400&#8211;1,250 m/z; then acquire up to 10 MS/MS spectra (100 ms each) with a mass range of 100&#8211;1,600 m/z and a linear gradient of 2%&#8211;80% acetonitrile and 0.2% formic acid for 80 min at a flow rate of 300 nL/min.</li>
	<li>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 (&#62; 95% fidelity) is required.</li>
	<li>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.</li>
</ol>

<ol><li>Thommen, M., Holtkamp, W., Rodnina, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-translational+protein+folding%3A+progress+and+methods%5BTitle%5D)+AND+%22Current+Opinion+in+Structural+Biology%22%5BJournal%5D)">Co-translational protein folding: progress and methods.</a> Current Opinion in Structural Biology. 42, 83-89 (2017).</li>
<li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+protein+catabolism+in+atherosclerotic+vascular+inflammation%5BTitle%5D)+AND+%22Frontiers+in+Cardiovascular+Medicine%22%5BJournal%5D)">Defective protein catabolism in atherosclerotic vascular inflammation.</a> Frontiers in Cardiovascular Medicine. 4, 79(2017).</li>
<li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+calpain+proteolytic+systems+underlies+degenerative+vascular+disorders%5BTitle%5D)+AND+%22Journal+of+Atherosclerosis+and+Thrombosis%22%5BJournal%5D)">Dysregulation of calpain proteolytic systems underlies degenerative vascular disorders.</a> Journal of Atherosclerosis and Thrombosis. 25 (1), 1-15 (2018).</li>
<li>Steckelberg, A. L., Boehm, V., Gromadzka, A. M., Gehring, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CWC22+connects+pre-mRNA+splicing+and+exon+junction+complex+assembly%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">CWC22 connects pre-mRNA splicing and exon junction complex assembly.</a> Cell Reports. 2 (3), 454-461 (2012).</li>
<li>Turriziani, B., von Kriegsheim, A., Pennington, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-protein+interaction+detection+via+mass+spectrometry-based+proteomics%5BTitle%5D)+AND+%22Advances+in+Experimental+Medicine+and+Biology%22%5BJournal%5D)">Protein-protein interaction detection via mass spectrometry-based proteomics.</a> Advances in Experimental Medicine and Biology. 919, 383-396 (2016).</li>
<li>Obama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+modified+apolipoprotein+B-100+structures+formed+in+oxidized+low-density+lipoprotein+using+LC-MS%2FMS%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS.</a> Proteomics. 7 (13), 2132-2141 (2007).</li>
<li>Matsudaira, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+from+picomole+quantities+of+proteins+electroblotted+onto+polyvinylidene+difluoride+membranes%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.</a> The Journal of Biological Chemistry. 262 (21), 10035-10038 (1987).</li>
<li>Yamaguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+method+for+N-terminal+sequencing+of+proteins+by+MALDI+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.</a> Analytical Chemistry. 77 (2), 645-651 (2005).</li>
<li>Iwamatsu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S-carboxymethylation+of+proteins+transferred+onto+polyvinylidene+difluoride+membranes+followed+by+in+situ+protease+digestion+and+amino+acid+microsequencing%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">S-carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing.</a> Electrophoresis. 13 (3), 142-147 (1992).</li>
<li>Strader, M. B., Tabb, D. L., Hervey, W. J., Pan, C., Hurst, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+specific+trypsin+digestion+of+microgram+to+nanogram+quantities+of+proteins+in+organic-aqueous+solvent+systems%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems.</a> Analytical Chemistry. 78 (1), 125-134 (2006).</li>
<li>Miyazaki, T., Miyazaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+roles+of+calpain+proteolytic+systems+in+macrophage+cholesterol+handling%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.</a> Cellular and Molecular Life Sciences. 74 (16), 3011-3021 (2017).</li>
<li>Miyazaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calpain-6+confers+atherogenicity+to+macrophages+by+dysregulating+pre-mRNA+splicing%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing.</a> Journal of Clinical Investigation. 126 (9), 3417-3432 (2016).</li>
<li>Tonami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calpain-6%2C+a+microtubule-stabilizing+protein%2C+regulates+Rac1+activity+and+cell+motility+through+interaction+with+GEF-H1%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Calpain-6, a microtubule-stabilizing protein, regulates Rac1 activity and cell motility through interaction with GEF-H1.</a> Journal of Cell Science. 124 (Pt 8), 1214-1223 (2011).</li>
<li>Rodnina, M. V., Wintermeyer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+elongation%2C+co-translational+folding+and+targeting%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Protein elongation, co-translational folding and targeting.</a> Journal of Molecular Biology. 428 (10 Pt B), 2165-2185 (2016).</li>
<li>Bunai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+analysis+of+acrylamide+gel+separated+proteins+immobilized+on+polyvinylidene+difluoride+membranes+following+proteolytic+digestion+in+the+presence+of+80%25+acetonitrile%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile.</a> Proteomics. 3 (9), 1738-1749 (2003).</li>
</ol>

The authors have nothing to disclose.

We have previously described an analysis of the oxidative modifications of apolipoprotein B-100 in oxidized low-density lipoprotein using LC-MS/MS preceded by an on-membrane digestion technique6. In the present study, we combined this technique with immunoprecipitation and have identified several calpain-6-associated proteins. This novel technique represents a convenient method of screening for candidate associated proteins. Calpain-6 is a non-proteolytic member of the calpain proteolytic family<s...

Although proteins play constitutive roles in living organisms, they are continually synthesized, processed, and degraded in the intracellular environment. Furthermore, intracellular proteins frequently physically and biochemically interact, which affects the function of one or both1,2,3. For example, the direct binding of spliceosome-associated protein homolog CWC22 with eukaryotic translation initiation factor 4A3 (eIF4A3) is necessary for the assembly of the exon junction complex4. Consistent with this, an eIF4A3 mutant that lacks affinity for CWC22 fails to facilitate exon junction complex-driven mRNA splicing4. Thus, the study of protein interactions is crucial for the precise understanding of physiologic regulation as well as of cellular functions.
Recent advances in mass spectrometry (MS) have been applied to the comprehensive analysis of protein-protein interactions. For instance, the co-precipitation of endogenous proteins or exogenously introduced tagged proteins with their associated proteins, followed by MS analysis, enables the identification of novel protein interactions5. However, one major bottleneck of MS/MS analysis is poor recovery from tryptic digests of protein samples. For conducting proteomic analyses on cell lysates, in-gel and on-membrane digestion techniques are generally employed to prepare MS/MS samples. We have previously compared an in-gel digestion procedure with an on-membrane digestion technique6, and showed that the latter was associated with better sequence coverage. Polyvinylidene difluoride (PVDF) membrane may be suitable for this purpose because it is mechanically robust and resistant to high concentrations of organic solvents7,8, permitting the enzymatic digestion of immobilized proteins in the presence of 80% acetonitrile9. Furthermore, immobilization on a membrane can induce conformational changes in target proteins, leading to improvements in tryptic digestion efficiency10. Accordingly, in this article, we have described the use of immunoprecipitation-LC/MS/MS analysis of protein interactions using an on-membrane digestion technique. This simple method facilitates the convenient analysis of protein-protein interactions even in non-specialist laboratories.

<table><tbody><tr><td>Acetonitrile</td><td>Wako</td><td>014-00386</td><td></td></tr><tr><td>Citric acid</td><td>Wako</td><td>030-05525</td><td></td></tr><tr><td>DiNA</td><td>KYA Tech Co.</td><td></td><td>nanoflow high-performance liquid chromatography</td></tr><tr><td>DiNa AI</td><td>KYA Tech Co.</td><td></td><td>nanoflow high-performance liquid chromatography equipped with autosampler</td></tr><tr><td>DTT</td><td>Nacalai tesque</td><td>14112-94</td><td></td></tr><tr><td>Dynabeads protein G</td><td>Thermo Fisher Scientific</td><td>10003D</td><td></td></tr><tr><td>Formic acid</td><td>Wako</td><td>066-00461</td><td></td></tr><tr><td>HiQ Sil C18W-3</td><td>KYA Tech Co.</td><td>E03-100-100</td><td>0.10mmID * 100mmL</td></tr><tr><td>Iodoacetamide</td><td>Wako</td><td>095-02151</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>L3000008</td><td></td></tr><tr><td>Living Colors A.v. Monoclonal Antibody (JL-8)</td><td>Clontech</td><td>632380</td><td></td></tr><tr><td>NaCl</td><td>Wako</td><td>191-01665</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>018-21742</td><td></td></tr><tr><td>Nonidet P-40</td><td>Sigma</td><td>N6507</td><td>poly(oxyethelene) octylphenyl ether (n=9)</td></tr><tr><td>peptide standard</td><td>KYA Tech Co.</td><td>tBSA-04</td><td>tryptic digests of bovine serum albumin</td></tr><tr><td>PP vial</td><td>KYA Tech Co.</td><td>03100S</td><td>plastic sample tube</td></tr><tr><td>Protease inhibitor cooctail</td><td>Sigma</td><td>P8465</td><td></td></tr><tr><td>ProteinPilot software</td><td>Sciex</td><td>5034057</td><td>software for protein identification</td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td>trypsin</td></tr><tr><td>Sodium orthovanadate</td><td>Sigma</td><td>S6508</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate</td><td>Sigma</td><td>71643</td><td></td></tr><tr><td>TFA</td><td>Wako</td><td>206-10731</td><td></td></tr><tr><td>trap column</td><td>KYA Tech Co.</td><td>A03-05-001</td><td>0.5mmID * 1mmL</td></tr><tr><td>TripleTOF 5600 system</td><td>Sciex</td><td>4466015</td><td>Hybrid quadrupole time-of-flight tandem mass spectrometer</td></tr><tr><td>Tris</td><td>Wako</td><td>207-06275</td><td></td></tr><tr><td>Tween-20</td><td>Wako</td><td>160-21211</td><td></td></tr></tbody></table>

evaluation of protein&#8211;protein interactions using an on-membrane digestion technique

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

By means of the above-described procedure, immunoprecipitants were analyzed using LC-MS/MS (Figure 1). After the exclusion of exogenously derived proteins (proteins from other species and IgGs), 17 proteins were identified in calpain-6-associated immunoprecipitants (Table 1) and 15 proteins were identified in GFP-associated immunoprecipitants (Table 2). Of the calpain-6 and GFP-associated proteins, 11 were identified in both ...

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

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