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

The repeatability of peptide identifications in shotgun proteome analyses employing strong cation‐exchange‐×ion‐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%.