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>

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	<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. 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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

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&#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.

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

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6.5K Views.  Showa University School of Pharmacy. 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–protein interactions.

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

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Bioengineering

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Source: Obama, T. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/59733">Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.</a>&#160;J. Vis. Exp.&#160; (2019)
This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Trawienie białek na błonie w celu przygotowania białek współimmunoprecypitowanych do badań interakcji

Source: Obama, T. et al., Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.&#160;J. Vis. Exp.&#160; (2019)
This ...

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

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Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Glycopeptide Capture for Cell Surface Proteomics

Trawienie białek na błonie w celu przygotowania białek współimmunoprecypitowanych do badań interakcji