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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 used to analyze protein-protein interactions. In order to facilitate the identification of co-immunoprecipitated proteins, mass spectrometry is often utilized^5,6,7,8,9. By targeting a known member of a protein complex with an antibody, it is possible to isolate the protein complex and to subsequently identify its unknown components via mass spectrometry analysis. ARIP4 (androgen receptor-interacting protein 4), a transcriptional coregulator, interacts with nuclear receptor proteins in order to activate or repress its target promoters in a context-dependent manner^9,10. To better understand the transcriptional regulatory mechanisms governing these nuclear factors, we describe a comprehensive method to purify and identify ARIP4 interacting proteins by using the LC-MS/MS system.

Access restricted. Please log in or start a trial to view this content.

Protokół

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 with ice-cold PBS and harvest the cells using a scraper. Transfer the cells into a 1.5 mltube and centrifuge it at 8,000 x g for 2 min at 4 °C. Aspirate the supernatant and resuspend the cell pellet in 5x the pellet volume of 0.4 M KCl buffer (High salt buffer: 20 mM Tris-HCl (pH 8.0), 0.4 M KCl, 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 10 mM β-mercaptethanol, 1x protease inhibitor cocktail). Rotate the mixture for 20 min at 4 °C.
2. Centrifuge the tube at 17,700 x g for 10 min at 4 °C.
3. Transfer the supernatant (whole-cell extract) to a new 2-mL tube and add 3x the initial volume of 0 M KCl buffer (Dilution buffer: 20 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 10 mM β-Mercaptoethanol, 1x protease inhibitor cocktail).
4. Perform another centrifugation (17,700 x g for 10 min at 4 °C) and transfer the supernatant (whole-cell extract) to a new 2 ml tube.

3. Immunoprecipitation

1. During the centrifugation (2.4), wash 50 µl of anti-FLAG beads with PBS, 0.1 M glycine-hydrochloride, and then 1 M Tris-hydrochloride, followed by washing twice with 0.1M KCl buffer (Low salt buffer: 20 mM Tris-HCl (pH 8.0), 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 10 mM β-mercaptoethanol, 1x protease inhibitor cocktail). The centrifugation for this washing process is performed at 2,000 x g for 1 min at 4 °C.
2. Mix 50 µl of FLAG beads with the whole-cell extracts and gently rotate the mixture for 4 hr at 2-4 °C (1% of the whole-cell extract from step 2.4 should also be kept at -80 °C for future use as an input).
3. Transfer the sample to a micro spin column (gravity drop). Keep the flow-through in a 1.5 ml tube, freeze it using liquid nitrogen, and store it at -80 °C (check the protein levels of both the input and flow-through using Western blot analysis).
4. Add 1 ml of 0.1 M KCl buffer (Low salt buffer) to wash the FLAG beads (gravity drop).
5. Repeat step 3.4 at least four more times (for a total of five washes).

4. Elution

1. Place the column into a 1.5 ml tube and centrifuge it at 2,000 x g for 1 min; the beads will dry.
2. Cap the bottom of the column.
3. Add 50 µl (equal to the volume of FLAG beads) of 0.1 M glycine-HCl (pH 2.5).
4. Gently shake the mixture for 10 min at room temperature.
5. Place the column into a new 1.5 ml tube and centrifuge it at 2,000 x g for 1 min.
6. The eluted solution is neutralized with 10 µl of 1 M Tris-HCl (pH 8.0); mix well by pipetting or by using a vortex machine.

5. Alkylation and Trypsinization

1. Add 50 µl of 100 mM NH₄HCO₃, 10 µl of CH₃CN, and 2 µl of dithiothreitol to the mixture (112 µl total volume).
2. Incubate the sample for 30 min at 56 °C.
3. Place the sample at room temperature for 10 min.
4. Add 10 µl of 333 mM iodoacetamide to the sample and incubate it in the dark for 30 min at 37 °C.
5. Add 10 µl of trypsin (33 ng/µl) to the mixture and incubate it overnight at 37 °C.

6. LC-MS/MS Analysis

1. Following overnight trypsinization, transport the final product to an LC-MS/MS facility or to a third-party mass spectrometry lab for analysis¹².

Access restricted. Please log in or start a trial to view this content.

Wyniki

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

Access restricted. Please log in or start a trial to view this content.

Dyskusje

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...

Access restricted. Please log in or start a trial to view this content.

Materiały

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