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Repeatability of peptide identifications in shotgun proteome analysis employing off-line two-dimensional chromatographic separations and ion-trap MS.

Authors
Type
Published Article
Journal
Journal of Separation Science
1615-9314
Publisher
Wiley Blackwell (John Wiley & Sons)
Publication Date
Volume
32
Issue
8
Pages
1156–1164
Identifiers
DOI: 10.1002/jssc.200800615
PMID: 19360783
Source
Medline
License
Unknown

Abstract

The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.

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