Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes.
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.
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc, NCI-Frederick, Frederick, Maryland 21702, United States.
Sudipto
S
Das
Allen D
AD
Bosley
Xiaoying
X
Ye
King C
KC
Chan
Isabel
I
Chu
Jeffery E
JE
Green
Haleem J
HJ
Issaq
Timothy D
TD
Veenstra
Thorkell
T
Andresson
D002412:Cations; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D050990:GATA3 Transcription Factor / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D014162:Transfection
C069077:BMI1 protein, human; D002412:Cations; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1