Comparison of Strong Cation Exchange and SDS-PAGE Fractionation for Analysis of Multiprotein Complexes

• Published in: Journal of proteome research 2010-12, Vol.9 (12), p.6696-6704
• Main Authors: Das, Sudipto, Bosley, Allen D., Ye, Xiaoying, Chan, King C., Chu, Isabel, Green, Jeffery E., Issaq, Haleem J., Veenstra, Timothy D., Andresson, Thorkell
• Format: Article
• Language: English
• Subjects: Cations; Cell Line, Tumor; Chemical Fractionation - methods; Chromatography, Ion Exchange - methods; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel - methods; GATA3 Transcription Factor - analysis; GATA3 Transcription Factor - genetics; HEK293 Cells; Humans; Mass Spectrometry; Multiprotein Complexes - analysis; Nuclear Proteins - analysis; Nuclear Proteins - genetics; Polycomb Repressive Complex 1; Proto-Oncogene Proteins - analysis; Proto-Oncogene Proteins - genetics; Repressor Proteins - analysis; Repressor Proteins - genetics; Reproducibility of Results; Transfection
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/pr100843x

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC−MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC−MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC−MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC−MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.