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Absolute quantification of the glycolytic pathway in yeast : deployment of a complete qconCAT approach
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Carroll, Kathleen M., Simpson, Deborah M., Eyers, Claire E., Knight, Christopher G., Brownridge, Philip, Dunn, Warwick B., Winder, Catherine L., Lanthaler, Karin, Pir, Pınar, Malys, Naglis, Kell, Douglas B., Oliver, Stephen G., Gaskell, Simon J. and Beynon, Robert J. (2011) Absolute quantification of the glycolytic pathway in yeast : deployment of a complete qconCAT approach. Molecular & Cellular Proteomics, 10 (12). M111.007633. doi:10.1074/mcp.M111.007633 ISSN 1535-9476.
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Official URL: http://dx.doi.org/10.1074/mcp.M111.007633
Abstract
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 LCMS, using ultra high performance liquid chromatographycoupled 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.
Item Type: | Journal Article | ||||||||||||||||||||||||
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Subjects: | Q Science > QK Botany | ||||||||||||||||||||||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||||||||||||||||||
Library of Congress Subject Headings (LCSH): | Yeast fungi| -- Genetics., Saccharomyces cerevisiae., Glycolysis., Enzymes., Yeast fungi -- Biotechnology. | ||||||||||||||||||||||||
Journal or Publication Title: | Molecular & Cellular Proteomics | ||||||||||||||||||||||||
Publisher: | American Society for Biochemistry and Molecular Biology | ||||||||||||||||||||||||
ISSN: | 1535-9476 | ||||||||||||||||||||||||
Official Date: | 1 December 2011 | ||||||||||||||||||||||||
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Volume: | 10 | ||||||||||||||||||||||||
Number: | 12 | ||||||||||||||||||||||||
Article Number: | M111.007633 | ||||||||||||||||||||||||
DOI: | 10.1074/mcp.M111.007633 | ||||||||||||||||||||||||
Status: | Peer Reviewed | ||||||||||||||||||||||||
Publication Status: | Published | ||||||||||||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||||||||||||||||||||
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