Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-mass spectrometry

Abstract

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress. We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.

Fig. 1.

Generation and use of MS/MS spectral libraries and SWATH- bioinformatic tools for the detection and quantification of proteomes and proteins. First, MS coordinates were selected from spectral libraries built from MS/MS spectra acquired by extensive data-dependent analysis experiments of fractionated protein digests. Second, those suitable MS/MS coordinates were used to trigger the fragment ion chromatogram extraction for the peptides of interest in the SWATH maps. Finally, the extracted chromatograms were scored using the probabilistic scoring model of mProphet and protein significance analysis was performed by MSstats.

Fig. 2.

SWATH-MS analysis of an unfractionated yeast cell lysate. A, Number of peptides per protein identified in a yeast cell digest. B, Proteins are spanning a concentration range of four orders of magnitude from 1e⁶ to 100 copies/cell according to quantitative Western blot analysis. C, Distribution of the cellular abundances (copies/cell) of the detected proteins according to quantitative Western blot analysis (34).

Fig. 3.

Consistency and reproducibility of SWATH measurements across four injections of an unfractionated tryptic yeast digest. A, Number of missing assays, peptides, and proteins detected across four technical replicates. B, Histogram of CVs of assays detected across all replicates. C, CVs of the assays detected across the entire abundance range and all replicates.

Fig. 4.

Accuracy of SWATH measurements. Estimated fold changes for all the proteins detected in both ¹⁴N- and ¹⁵N-labeled backgrounds (i.e. 1:1; ¹⁴N:¹⁵N and 1:1/10; ¹⁴N:¹⁵N) and all biological replicates. The first box is the estimated fold change of 771 proteins from the study with expected fold change equal to one (i.e. 1:1; ¹⁴N:¹⁵N). The second box is the estimated fold change of 502 proteins from the study with expected fold change equal to 10 (i.e. 1:1/10; ¹⁴N:¹⁵N). The red line in box 1 indicates a fold change of one. The red line in box 2 indicates a fold change of 10.

Fig. 5.

Hierarchical clustering of significantly changing proteins after osmotic shock by SWATH-MS. The osmotic shock was applied by adding 0.4 m NaCl to each 50 ml culture and after 0 min (T0), 15 min (T1), 30 min (T2), 60 min (T3), 90 min (T4), and 120 min (T5) the cells were harvested and proteins were isolated and digested. A, In the heatmap, significant up-regulation shown in red, significant down-regulation is shown in blue, and no statistically significant change in abundance is shown in yellow. Color intensity reflects the corresponding log₂ fold change. Four main clusters of proteins, according to their patterns of differential abundance in time as compared with the baseline time 0 min (T0) are represented. B, Expression profile patterns of proteins belonging to the four clusters, where all the time points (i.e. T1, T2, T3, T4, and T5) were compared with the time point 0 min (T0).

Fig. 6.

Comparison between proteomics and transcripts data sets. Heat maps for each transcript (top raw) and the corresponding protein log₂-fold changes (bottom raw) between 30 min (T2), 60 min (T3), 90 min (T4), 120 min (T5), and the initial time point 0 min (T0) together with the distribution of Pearson correlation coefficients calculated for all detected protein-transcript pairs in A, Glycolysis-gluconeogenesis pathway, B, Pentose phosphate pathway, C, Glycine, serine and threonine metabolism pathway, and D, Phenylalanine, tyrosine, and tryptophan pathway. Significant up-regulation is shown in red, significant down-regulation in blue, and no statistically significant change in abundance is shown in yellow. Color intensity reflects the corresponding log₂ fold change.