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Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing‐based proteomic platform coupled with a spectral counting approach
Author(s) -
Balgley Brian M.,
Wang Weijie,
Song Tao,
Fang Xueping,
Yang Li,
Lee Cheng S.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800050
Subject(s) - proteome , reproducibility , isoelectric focusing , proteomics , chromatography , quantitative proteomics , chemistry , shotgun proteomics , shotgun , mass spectrometry , biochemistry , enzyme , gene
Multidimensional separations of the peptides resulting from enzymatic digestions of complex protein mixtures prior to MS/MS, namely shotgun proteomics, is increasingly utilized for large‐scale identification and quantitation of proteins. Inherent to the performance of proteomic measurements is the resolving power of each of the separations both separately and in combination. By simply raising the number of CIEF fractions, the resulting enhancement in the overall peak capacity of combined CIEF/nano‐RPLC separations greatly reduces the complexity of eluted peptides prior to MS detection and sequencing and increases the proteome coverage. The capabilities of the CIEF‐based proteome platform coupled with the spectral counting approach to confidently and reproducibly quantify proteins and changes in protein expression levels among samples are evaluated. Analytical reproducibility of relative protein abundance is determined to exhibit a Pearson R 2 value greater than 0.99 and a CV of 14.1%. The platform is demonstrated to be capable of measuring changes in protein expression as low as 1.5‐fold, with confidence following multiple testing adjustment.