A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Summary

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Abstract

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

Introduction

Protein-protein interactions play an important role in many biological functions. As such, these interactions have been implicated in signal transduction; protein transport across cell membranes; cell metabolism; and several nuclear processes, including DNA replication, DNA damage repair, recombination, and transcription^1,2,3,4. Identifying the proteins involved in these interactions is therefore critical to advancing our understanding of these cellular processes.

Immunoprecipitation (IP) is a validated technique ....

Protocol

1. Transfection

1. Seed HEK293 cells in 100-mm culture plates (2 x 10⁶ cells/dish). Culture the cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. Incubate the cells in a humidified incubator at 5% CO₂ and 37 °C.
2. The next day, transfect the cells with 10 µg of FLAG-tagged ARIP4 plasmid using 40 µl of transfection reagent according to the manufacturer's directions¹¹.

2. Protein Extraction

1. Approximately 36 hr post-transfection, wash the cells w....

Results

We identified a strong ARIP4 signal, around 160 KDa, as well as those of a mock sample control and several other unknown proteins (Figure 1). LC-MS/MS analysis identified both the ARIP4 complex peptides and the potential peptide cofactors within the fraction of FLAG beads (Table 1). p62 (Sequestosome1), a known ARIP4 cofactor¹¹ was identified in the analysis (Table 1), which confirms the efficacy of this system

Discussion

Efficient transfection is essential to obtain a successful result with this protocol. Accordingly, we recommend using Western blot analysis to determine the immunoprecipitated FLAG-tagged protein levels. This step enables users to verify that their protein of interest is properly overexpressed and, moreover, that the IP was performed successfully. FLAG-tagged protein levels should also be checked prior to the mass spectrometry analysis.

A mock sample should be used as a negative control to avo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipofectamine 2000 Transfection Reagent	Thermo Fisher Scientific	11668019
Protease Inhibitor Cocktail (EDTA free) (100x)	nacalai tesque	03969-21
ANTI-FLAG M2 Affinity Gel	Sigma-Aldrich	A2220
Micro Bio-Spin Columns	BIO-RAD	732-6204