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Repeatability of peptide identifications in shotgun proteome analysis employing off‐line two‐dimensional chromatographic separations and ion‐trap MS
Author(s) -
Delmotte Nathanaël,
Lasaosa Maria,
Tholey Andreas,
Heinzle Elmar,
van Dorsselaer Alain,
Huber Christian G.
Publication year - 2009
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200800615
Subject(s) - repeatability , chromatography , shotgun , chemistry , shotgun proteomics , peptide , proteome , ion trap , mass spectrometry , analytical chemistry (journal) , proteomics , biochemistry , gene
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%.