A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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 ....
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.
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 .......
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
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).
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved