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Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy
Author(s) -
Brownridge Philip,
Holman Stephen W.,
Gaskell Simon J.,
Grant Christopher M.,
Harman Victoria M.,
Hubbard Simon J.,
Lanthaler Karin,
Lawless Craig,
O'cualain Ronan,
Sims Paul,
Watkins Rachel,
Bey Robert J.
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100039
Subject(s) - proteome , isotopologue , isotope dilution , proteomics , chemistry , mass spectrometry , analyte , yeast , workflow , computational biology , quantitative proteomics , chromatography , saccharomyces cerevisiae , yield (engineering) , computer science , biochemistry , biology , gene , database , materials science , molecule , organic chemistry , metallurgy
In this paper, we discuss the challenge of large‐scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae . We have based our strategy on the well‐established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co‐digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.