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. 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>

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>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 ProteinÃ¢â‚¬â€œProtein 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

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

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 ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. 
In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

Here, we present an optimized on-filter digestion protocol with detailed information about the following: protein digestion, peptide purification and data independent acquisition analysis. This strategy is applied to the analysis of expressed prostatic secretions-urine samples and allows high proteome coverage and low missing value label-free profiling of the urinary proteome.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...