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 x 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%.