Repeatability of peptide identifications in shotgun proteome analysis employing off-line two-dimensional chromatographic separations and ion-trap MS
✍ Scribed by Nathanaël Delmotte; Maria Lasaosa; Andreas Tholey; Elmar Heinzle; Alain van Dorsselaer; Christian G. Huber
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 564 KB
- Volume
- 32
- Category
- Article
- ISSN
- 1615-9306
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✦ Synopsis
Abstract
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%.