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Multi‐dimensional HPLC/MS of the nucleolar proteome using HPLC‐chip/MS
Author(s) -
Vollmer Martin,
Hörth Patric,
Rozing Gerard,
Couté Yohann,
Grimm Rudi,
Hochstrasser Denis,
Sanchez JeanCharles
Publication year - 2006
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.200500334
Subject(s) - high performance liquid chromatography , chromatography , chemistry , proteome , mass spectrometry , biochemistry
The proteome of the human nucleolus was investigated in a single analysis using off‐line strong cation exchange chromatography and microfraction collection combined with HPLC‐chip/MS. The analysis was conducted either as a 1‐D workflow with HPLC‐chip alone or as a 2‐D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2‐D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1‐D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false‐positive identifications, were applied for reliable protein identification.