Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.
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 microg 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 (pI) units, which make it possible to analyze organisms with significantly larger genomes/proteomes. Accurate pI 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 pI prediction can be evaluated as a function of their position in the peptide chain.
Cargile
Benjamin J
BJ
Mass Spectrometry Program, Research Triangle Institute, 3040 Conrwallis Road, Research Triangle Park, NC 27709-2194, USA.
Talley
Dana L
DL
Stephenson
James L
JL
D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D018892:Proton-Motive Force
D010455:Peptides; D011506:Proteins