High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification.
Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.
Tu
Cheng-Jian
CJ
Research Center for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China.
Dai
Jie
J
Li
Su-Jun
SJ
Sheng
Quan-Hu
QH
Deng
Wen-Jun
WJ
Xia
Qi-Chang
QC
Zeng
Rong
R
D000328:Adult; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis*; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*
D001798:Blood Proteins; D020543:Proteome