Title Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach /
Authors Carroll, Kathleen M ; Simpson, Deborah M ; Eyers, Claire E ; Knight, Christopher G ; Brownridge, Philip ; Dunn, Warwick B ; Winder, Catherine L ; Lanthaler, Karin ; Pir, Pinar ; Malys, Naglis ; Kell, Douglas B ; Oliver, Stephen G ; Gaskell, Simon J ; Beynon, Robert J
DOI 10.1074/mcp.M111.007633
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Is Part of Molecular & cellular proteomics.. Bethesda, MD : American Society for Biochemistry and Molecular Biology. 2011, vol. 10, iss. 12, art. no. M111.007633, p. 1-15.. ISSN 1535-9476. eISSN 1535-9484
Keywords [eng] concatenated signature peptides ; mass-spectrometry ; protein identification ; saccharomyces-cerevisiae ; quantitative-analysis ; shotgun proteomics ; bicinchoninic acid ; sequence databases ; lc-ms/ms ; technology
Abstract [eng] The availability of label-free data derived from yeast cells (based on the summed intensity of the three strongest, isoform-specific peptides) permitted a preliminary assessment of protein abundances for glycolytic proteins. Following this analysis, we demonstrate successful application of the QconCAT technology, which uses recombinant DNA techniques to generate artificial concatamers of large numbers of internal standard peptides, to the quantification of enzymes of the glycolysis pathway in the yeast Saccharomyces cerevisiae. A QconCAT of 88 kDa (59 tryptic peptides) corresponding to 27 isoenzymes was designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by LC-MS, using ultra high performance liquid chromatography-coupled mass spectrometry. We were able to determine absolute protein concentrations between 14,000 and 10 million molecules/cell. Issues such as efficiency of extraction and completeness of proteolysis are addressed, as well as generic factors such as optimal quantotypic peptide selection and expression. In addition, the same proteins were quantified by intensity-based label-free analysis, and both sets of data were compared with other quantification methods.
Published Bethesda, MD : American Society for Biochemistry and Molecular Biology
Type Journal article
Language English
Publication date 2011
CC license CC license description