Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides |
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Authors: | Cargile Benjamin J Talley Dana L Stephenson James L |
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Affiliation: | Mass Spectrometry Program, Research Triangle Institute, 3040 Conrwallis Road, Research Triangle Park, NC 27709-2194, USA. |
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Abstract: | 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. |
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Keywords: | Immobilized pH gradient Isoelectric focusing Mass spectrometry Peptide Protein identification Proteomics |
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