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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein–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–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–protein interactions.

Introduction

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 ....

Protocol

1. Immunoprecipitation

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

  1. Transfect cultured cells with vectors encoding an epitope tag alone or a fusion protein. For acquiring representative data, transfer J774 cells (1 x 106) with vectors encoding green fluorescent protein .......

Representative Results

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 .......

Discussion

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

Acknowledgements

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).

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Materials

NameCompanyCatalog NumberComments
AcetonitrileWako014-00386
Citric acidWako030-05525
DiNAKYA Tech Co.nanoflow high-performance liquid chromatography
DiNa AIKYA Tech Co.nanoflow high-performance liquid chromatography equipped with autosampler
DTTNacalai tesque14112-94
Dynabeads protein GThermo Fisher Scientific10003D
Formic acidWako066-00461
HiQ Sil C18W-3KYA Tech Co.E03-100-1000.10mmID * 100mmL
IodoacetamideWako095-02151
Lipofectamine 3000Thermo Fisher ScientificL3000008
Living Colors A.v. Monoclonal Antibody (JL-8)Clontech632380
NaClWako191-01665
NH4HCO3Wako018-21742
Nonidet P-40SigmaN6507poly(oxyethelene) octylphenyl ether (n=9)
peptide standardKYA Tech Co.tBSA-04tryptic digests of bovine serum albumin
PP vialKYA Tech Co.03100Splastic sample tube
Protease inhibitor cooctailSigmaP8465
ProteinPilot softwareSciex5034057software for protein identification
Sequencing Grade Modified TrypsinPromegaV5111trypsin
Sodium orthovanadateSigmaS6508
Sodium phosphate dibasic dihydrateSigma71643
TFAWako206-10731
trap columnKYA Tech Co.A03-05-0010.5mmID * 1mmL
TripleTOF 5600 systemSciex4466015Hybrid quadrupole time-of-flight tandem mass spectrometer
TrisWako207-06275
Tween-20Wako160-21211

References

  1. Thommen, M., Holtkamp, W., Rodnina, M. V. Co-translational protein folding: progress and methods. Current Opinion in Structural Biology. 42, 83-89 (2017).
  2. Miyazaki, T., Miyazaki, A. Defective protein catabolism in atheroscleroti....

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Protein protein InteractionOn membrane DigestionProteomic AnalysisImmunoprecipitationMagnetic BeadsPVDF MembraneMass Spectrometry

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