期刊
ELECTROPHORESIS
卷 25, 期 6, 页码 936-945出版社
WILEY
DOI: 10.1002/elps.200305722
关键词
immobilized pH gradient; isoelectric focusing; mass spectrometry; peptide; protein identification; proteomics
In this work, we demonstrate the potential use of immobilized pH gradient isoelectric focusing as a first dimension in shotgun proteomics. The high resolving power and resulting reduction in matrix ionization effects due to analyzing peptides with almost the exact same physiochemical properties, represents a significant improvement in performance over traditional strong cation-exchange first-dimensional analysis associated with the shotgun proteomics approach. For example, using this technology, we were able to identify more than 6000 peptides and > 1200 proteins from the cytosolic fraction of Escherichia coli from approximately 10 mug of material analyzed in the second-dimensional liquid chromatography-tandem mass spectrometry experiment. Sample loads on the order of 1 mg can be resolved to 0.25 isoelectric point (pl) units, which make it possible to analyze organisms with significantly larger genomes/proteomes. Accurate pl prediction can then be employed using currently available algorithms to very effectively filter data for peptide/protein identification, and thus lowering the false-positive rate for cross-correlation-based peptide identification algorithms. By simplifying the protein mixture problem to tryptic peptides, the effect of specific amino acids on pl prediction can be evaluated as a function of their position in the peptide chain.
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