Relative and Absolute Quantitation in Mass Spectrometry–Based Proteomics

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.

We report a method for large-scale absolute protein expression measurements (APEX) and apply it to estimate the relative contributions of transcriptional- and translational-level gene regulation in the yeast and Escherichia coli proteomes. APEX relies upon correcting each protein's mass spectrometry sampling depth (observed peptide count) by learned probabilities for identifying the peptides. APEX abundances agree with measurements from controls, western blotting, flow cytometry and two-dimensional gels, as well as known correlations with mRNA abundances and codon bias, providing absolute protein concentrations across approximately three to four orders of magnitude. Using APEX, we demonstrate that 73% of the variance in yeast protein abundance (47% in E. coli) is explained by mRNA abundance, with the number of proteins per mRNA log-normally distributed about approximately 5,600 ( approximately 540 in E. coli) protein molecules/mRNA. Therefore, levels of both eukaryotic and prokaryotic proteins are set per mRNA molecule and independently of overall protein concentration, with >70% of yeast gene expression regulation occurring through mRNA-directed mechanisms.