title: Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach.
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 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.
authors.affiliation: Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom. Kathleen.Carroll@manchester.ac.uk
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Carroll
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Simpson
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Knight
authors.forename: Philip
authors.initials: P
authors.lastname: Brownridge
authors.forename: Warwick B
authors.initials: WB
authors.lastname: Dunn
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Winder
authors.forename: Karin
authors.initials: K
authors.lastname: Lanthaler
authors.forename: Pinar
authors.initials: P
authors.lastname: Pir
authors.forename: Naglis
authors.initials: N
authors.lastname: Malys
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Kell
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Oliver
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gaskell
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Beynon
mesh_terms: D000595:Amino Acid Sequence; D015870:Gene Expression; D006019:Glycolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000592:standards; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics; D012015:Reference Standards; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins