Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification.
Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.
Li
Rong-Xia
RX
Research Center for Proteome Analysis, Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
Zhou
Hu
H
Li
Su-Jun
SJ
Sheng
Quan-Hu
QH
Xia
Qi-Chang
QC
Zeng
Rong
R
D002853:Chromatography, Liquid / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000145:classification / Q000302:isolation & purification
D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins