A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay.

Andrew J Woo,Evelyn Susanto,Daniela Ulgiati,Lawrence J Abraham,James S Dods

doi:10.1074/mcp.t200003-mcp200

Abstract

Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression. Traditionally, the isolation and identification of transcription factors has been a long and laborious task. We present here a novel method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) using the power of two-dimensional electrophoresis coupled with mass spectrometry. By coupling SDS-PAGE and isoelectric focusing to EMSA, the molecular mass and pI of a protein complex seen in EMSA were estimated. Candidate proteins were then identified on a two-dimensional array at the predetermined pI and molecular mass coordinates and identified by mass spectrometry. We show here the successful isolation of a functionally relevant transcription factor and validate the identity through EMSA supershift analysis.

Highlights

Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression
We were interested in identifying the components of complex E that bound to this repeat sequence
We present a method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) that utilizes the power of 2DE, coupled with mass spectrometry

Summary

EXPERIMENTAL PROCEDURES

Nuclear Extract Preparation and S-300 Gel Filtration—Jurkat (T cell) cell lines were obtained from ATCC and maintained in RPMI 1640 medium with 2 mM L-glutamine, 100 ␮g/ml each of streptomycin and penicillin, and 10% fetal bovine serum at 37 °C with 5% CO2. Proteins were electrophoresed at 240 V on an 8% SDS-polyacrylamide gel [9], and the lane containing nuclear extract was sliced uniformly into molecular mass intervals. IEF Analysis—S-300 fractionated nuclear extract (180 ␮g) was resuspended in rehydration solution (8 M urea, 4% CHAPS, 0.5% IPG buffer, 2 mM tributyl phosphine) using ULTRAFREE centrifugal filters (Millipore). Nuclear proteins (2 ␮g) or pI/MM fractions (12.5 ␮l) were incubated for 10 min on ice with 1 ␮g of poly(dI1⁄7dC) in binding buffer consisting of 4% Ficoll, 20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl. Proteins were incubated with the 32P-labeled oligonucleotide for 30 min on ice prior to being loaded onto a 6% polyacrylamide gel containing 0.25ϫ Tris-taurine/EDTA. Monoisotopic peaks were searched against human proteins, 1–100 kDa, with a maximum of one missed cleavage, unmodified cysteines, and with a mass tolerance of 100 ppm

RESULTS

NCBI accession number

DISCUSSION