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

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 both^1,2,3. 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 .......

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

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Wako	014-00386
Citric acid	Wako	030-05525
DiNA	KYA Tech Co.		nanoflow high-performance liquid chromatography
DiNa AI	KYA Tech Co.		nanoflow high-performance liquid chromatography equipped with autosampler
DTT	Nacalai tesque	14112-94
Dynabeads protein G	Thermo Fisher Scientific	10003D
Formic acid	Wako	066-00461
HiQ Sil C18W-3	KYA Tech Co.	E03-100-100	0.10mmID * 100mmL
Iodoacetamide	Wako	095-02151
Lipofectamine 3000	Thermo Fisher Scientific	L3000008
Living Colors A.v. Monoclonal Antibody (JL-8)	Clontech	632380
NaCl	Wako	191-01665
NH4HCO3	Wako	018-21742
Nonidet P-40	Sigma	N6507	poly(oxyethelene) octylphenyl ether (n=9)
peptide standard	KYA Tech Co.	tBSA-04	tryptic digests of bovine serum albumin
PP vial	KYA Tech Co.	03100S	plastic sample tube
Protease inhibitor cooctail	Sigma	P8465
ProteinPilot software	Sciex	5034057	software for protein identification
Sequencing Grade Modified Trypsin	Promega	V5111	trypsin
Sodium orthovanadate	Sigma	S6508
Sodium phosphate dibasic dihydrate	Sigma	71643
TFA	Wako	206-10731
trap column	KYA Tech Co.	A03-05-001	0.5mmID * 1mmL
TripleTOF 5600 system	Sciex	4466015	Hybrid quadrupole time-of-flight tandem mass spectrometer
Tris	Wako	207-06275
Tween-20	Wako	160-21211