title: Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.
abstract: Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800Saccharomyces cerevisiaeproteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a "gold-standard" reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies.
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK;
authors.forename: Craig
authors.initials: C
authors.lastname: Lawless
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Holman
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Brownridge
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK;
authors.forename: Karin
authors.initials: K
authors.lastname: Lanthaler
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Harman
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK;
authors.forename: Rachel
authors.initials: R
authors.lastname: Watkins
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Dean E
authors.initials: DE
authors.lastname: Hammond
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Miller
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK;
authors.forename: Paul F G
authors.initials: PF
authors.lastname: Sims
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK;
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Grant
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
authors.affiliation: §Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Beynon
authors.affiliation: From the ‡Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK; simon.hubbard@manchester.ac.uk.
authors.forename: Simon J
authors.identifier: http://orcid.org/0000-0002-8601-9524
authors.initials: SJ
authors.lastname: Hubbard
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods; D007553:Isotope Labeling; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D040901:Proteomics / Q000379:methods* / Q000592:standards; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins