title: A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards.
abstract: The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance.
authors.affiliation: Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Kito
authors.affiliation: Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Okada
authors.affiliation: Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ishibashi
authors.affiliation: Department of Biochemistry, Kyushu University Graduate School of Medical Science, Fukuoka, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okada
authors.affiliation: Department of Biochemistry, Kyushu University Graduate School of Medical Science, Fukuoka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
mesh_terms: D004926:Escherichia coli; D005069:Evaluation Studies as Topic; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000592:standards*; D012015:Reference Standards; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin
keywords: Absolute quantification; Budding yeast; Mass spectrometry; Peptide-concatenated standard (PCS); Technology