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Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach
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 QconC...
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Published in: | Molecular & cellular proteomics 2011-12, Vol.10 (12), p.M111.007633-M111.007633 |
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container_title | Molecular & cellular proteomics |
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creator | 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 |
description | 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. |
doi_str_mv | 10.1074/mcp.M111.007633 |
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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. 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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. 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subjects | Amino Acid Sequence Concatamers Data processing DNA Enzymes Gene Expression Glycolysis Isoenzymes Isoenzymes - chemistry Isoenzymes - genetics Isoenzymes - metabolism Isotopes Mass spectroscopy Molecular Sequence Data Peptide Fragments - chemistry Peptide Fragments - standards Protein Processing, Post-Translational Proteolysis Proteomics Reference Standards Reproducibility of Results Saccharomyces cerevisiae Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - chemistry Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Tandem Mass Spectrometry - standards Tryptic peptides |
title | Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach |
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