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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.

SUBMITTER: Lawless C

PROVIDER: S-EPMC4824857 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

Lawless Craig C Holman Stephen W SW Brownridge Philip P Lanthaler Karin K Harman Victoria M VM Watkins Rachel R Hammond Dean E DE Miller Rebecca L RL Sims Paul F G PF Grant Christopher M CM Eyers Claire E CE Beynon Robert J RJ Hubbard Simon J SJ

Molecular & cellular proteomics : MCP 20160110 4

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 ...[more]

PMID: 26750110

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Project description:For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

Dataset Information

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

Publications

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

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