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 ...

<ol>
	<li>Thommen, M., Holtkamp, W., Rodnina, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+protein+folding:+progress+and+methods.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+protein+catabolism+in+atherosclerotic+vascular+inflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+calpain+proteolytic+systems+underlies+degenerative+vascular+disorders.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CWC22+connects+pre-mRNA+splicing+and+exon+junction+complex+assembly.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interaction+detection+via+mass+spectrometry-based+proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+modified+apolipoprotein+B-100+structures+formed+in+oxidized+low-density+lipoprotein+using+LC-MS/MS.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+from+picomole+quantities+of+proteins+electroblotted+onto+polyvinylidene+difluoride+membranes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+method+for+N-terminal+sequencing+of+proteins+by+MALDI+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-carboxymethylation+of+proteins+transferred+onto+polyvinylidene+difluoride+membranes+followed+by+in+situ+protease+digestion+and+amino+acid+microsequencing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+trypsin+digestion+of+microgram+to+nanogram+quantities+of+proteins+in+organic-aqueous+solvent+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+calpain+proteolytic+systems+in+macrophage+cholesterol+handling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-6+confers+atherogenicity+to+macrophages+by+dysregulating+pre-mRNA+splicing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-6,+a+microtubule-stabilizing+protein,+regulates+Rac1+activity+and+cell+motility+through+interaction+with+GEF-H1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+elongation,+co-translational+folding+and+targeting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+acrylamide+gel+separated+proteins+immobilized+on+polyvinylidene+difluoride+membranes+following+proteolytic+digestion+in+the+presence+of+80%+acetonitrile.">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>

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>NH4HCO3</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

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 ...

Watch this Scientific Journal Video about Evaluation of Proteinâ€“Protein Interactions using an On-Membrane Digestion Technique at JoVE.com

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

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.

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 ...

Researching protein-protein interaction is indispensable for understanding physiological processes in several field. In this video we introduce on-membrane digestion technique which allows the researchers to perform a comprehensive analysis of binding proteins by MS in a convenient manner. The main advantage of this technique is that we can improve the throughput of proteomic analysis for crude immunoprecipitants because separation of proteins by polyacrylamide gel electrophoresis is not necessary.Begin conjugating the antibody to the magnetic beads, by mixing the anti-GFP antibody with the beads in 500 microliters of citrate phosphate buffer. Rotate the mixture at 50 RPM for one hour at room temperature. And then wash the conjugated beads three times with citrate phosphate buffer containing 1%polysorbate 20.Lyse transfected cells according to manuscript directions. Scrape the dish and transfer the lysate into the 1.5 milliliter test tube. Then clear the lysate by centrifuging at 15, 000 x g for five minutes and collect the supernatant.Prepare unlabeled magnetic beads by washing them three times with 500 microliters of citrate phosphate buffer. After the final wash, remove the buffer and add cell lysate to the beads. Rotate the mixture at 50 RPM for 30 minutes at room temperature.Perform magnetic separation on the magnetic stand and collect the cell lysate. To bind the target proteins to the immunoglobulin conjugated beads, place the beads on the magnetic stand for five minutes. Discard the citrate buffer and add the cell lysate to the beads.Rotate the mixture for one hour at 50 RPM. To separate target proteins from the free non-target proteins, wash the beads three times with 500 microliters of citrate phosphate buffer containing 1%polysorbate 20. After the final wash, add 30 microliters of citrate buffer, and let the beads sit for five minutes at room temperature to elute the target proteins.To prepare the membrane, cut PVDF membranes into 3 millimeter by 3 millimeter pieces using surgical scissors that were cleaned immediately prior to use. Place the pieces of membrane on aluminum foil and add two to five microliters of ethanol to each piece. Before membranes are completely dry, add two to five microliters of the protein eluent to each piece and air dry them until the membrane surface becomes matte.Then transfer membranes into 1.5 milliliter tubes and store them at four degrees Celsius. If using immediately, add two to 30 microliters of ethanol which will make them hydrophilic. Remove the ethanol with a pipette, but before the membranes dry, completely add 200 microliters of DTT-based reaction solution to each tube, and incubate them at 56 degrees Celsius for one hour.After incubation, replace the reaction solution with 300 microliters of iodoacetamide solution, and incubate in the dark for 45 minutes. Then wash the membranes twice with distilled water and once with 2%acetonitrile by vortexing for at least ten seconds. Working with diaphanous-related acetonitrile can be extremely hazardous.A mask, glasses and gloves should always be worn when performing the on-membrane digestion procedure. Prepare trypsin according to manuscript directions, and add 100 microliters of trypsin reaction solution to each membrane. Incubate overnight at 37 degrees Celsius for digestion.The next day, transfer the reaction solution into a clean 1.5 milliliter tube, and add 100 microliters of wash solutions to the membrane. Incubate the membrane at 60 degrees Celsius for two hours, then collect the wash solution and mix it with the reaction solution. Add another 100 microliters of wash solution to the membrane and sonicate it for 10 minutes.Then collect the wash solution and mix it with the reaction solution. Cover the test tube with a piece of laboratory film and make small holes in the film with a needle. Dry the solution using a vacuum concentrator, and dissolve the residue in 10 microliters of 2%formic acid.Centrifuge the tube at 12, 000 x g for three minutes, and transfer the supernatant into a sample tube. Analyze the sample using a mass spectrometer linked to a nano LC/HPLC system. Using this protocol, 17 proteins were identified in the Calpain-6 associated immunoprecipitant, 15 in the GFP-associated immunoprecipitant, and 11 were identified in both.It's important to note that detection of at least one high fidelity peptide fragment is required for positive identification of a candidate protein. Proteins that precipitated in the GFP-expressing lysate as well as histones, exogenous and ribosomal proteins were omitted from the list. Washing the membranes before tryptic digestion with distilled water and 2%acetonitrile is the most important step in the procedure of on-membrane digestion over proteins.Following this method, it is possible to confirm co-precipitation of target protein by conducting other experiments, such as immunoprecipitation and Western blot analysis. This simple method allows the researchers to explore the novel protein-protein interactions with convenient and high-sensitive analysis.

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6.4K Views.  Showa University School of Pharmacy. Researching protein-protein interaction is indispensable for understanding physiological processes in several field. In this video we introduce on-membrane digestion technique which allows the researchers to perform a comprehensive analysis of binding proteins by MS in a convenient manner. The main advantage of this technique is that we can improve the throughput of proteomic analysis for crude immunoprecipitants because separation of proteins by polyacrylamide gel electrophoresis is not nece...