Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe

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Abstract
The fission yeast Schizosaccharomyces pombe is a widely used model organism to study basic mechanisms of eukaryotic biology, but unlike other model organisms, its proteome remains largely uncharacterized. Using a shotgun proteomics approach based on multidimensional prefractionation and tandem mass spectrometry, we have detected ∼30% of the theoretical fission yeast proteome. Applying statistical modelling to normalize spectral counts to the number of predicted tryptic peptides, we have performed label‐free quantification of 1465 proteins. The fission yeast protein data showed considerable correlations with mRNA levels and with the abundance of orthologous proteins in budding yeast. Functional pathway analysis indicated that the mRNA–protein correlation is strong for proteins involved in signalling and metabolic processes, but increasingly discordant for components of protein complexes, which clustered in groups with similar mRNA–protein ratios. Self‐organizing map clustering of large‐scale protein and mRNA data from fission and budding yeast revealed coordinate but not always concordant expression of components of functional pathways and protein complexes. This finding reaffirms at the protein level the considerable divergence in gene expression patterns of the two model organisms that was noticed in previous transcriptomic studies. ### Synopsis The unicellular archiascomycete fungus Schizosaccharomyces pombe is a well‐established model organism, but only ∼1500 of its predicted ∼4900 genes and proteins have been experimentally characterized. Weighing the advantages and disadvantages of currently available methods for quantitative proteomics, we have embarked on a mass spectrometry‐based approach for relative quantitation of native, unmodified fission yeast proteins. In addition, we have compared mRNA and protein expression profiles in fission yeast and budding yeast to assess the overall protein–mRNA correlation in these related organisms. We devised an extensive multidimensional biochemical prefractionation scheme of total cell lysate from wild‐type fission yeast cells, followed by analysis of individual fractions by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC ESI MS/MS). Approximately 3 million mass spectra were matched to 12 413 non‐redundant peptides, resulting in the identification of 1465 proteins (∼29.5% of the predicted fission yeast proteome). The list of proteins was representative of the whole proteome across the entire range of molecular weights, isoelectric points and gene ontology (GO) attributes. More detailed analysis revealed equal identification rates for essential and non‐essential proteins (both 36%). Similarly, yeast‐specific proteins were represented at the same rate as the entire proteome (30%). Metazoan ‘core’ proteins were overrepresented (47%), whereas we undersampled proteins containing predicted transmembrane domains (14%) and S. pombe ‐specific proteins (10%). To quantitatively rank the identified proteins relative to each other we used spectral counts ([Liu et al , 2004][1]; [Kislinger et al , 2006][2]). A negative binomial regression model was developed to adjust spectral counts to the number of predicted tryptic peptides allowing one miscleavage. Based on adjusted spectral counts (ASCs), we assembled an abundance ranked list of all 1465 proteins identified, which was validated by comparing it to absolute quantitation data established for a series of cytokinesis‐related fission yeast proteins ([Wu and Pollard, 2005][3]). Plotting our ASC data versus the absolute quantitation data revealed a close correlation ( r P=0.98), suggesting that ASCs provide a good approximation of relative protein abundance. The range of ASCs spanned more than three orders of magnitude. The mean ASC was 68.0, whereas the median was 14.6, indicating that the vast majority of the 1465 proteins identified are of relatively low abundance. The median of metazoan core proteins (ASC=24.2) is significantly higher than that of all proteins detected, whereas the abundance of S. pombe ‐specific proteins is considerably lower (ASC=5.5). In addition, essential proteins are considerably more abundant (median ASC=12.6) than non‐essential proteins (ASC=7.5). This finding can be rationalized by the enrichment of highly expressed core proteins in the set of essential proteins ([Supplementary Data File 2][4]). Analysis of 10 protein complexes for which we identified greater than 80% of their known or predicted subunits indicated that the protein synthesis machinery (ribosome, eIFs) and also the protein folding and degradation machinery (CCT chaperonin, proteasome) are among the most abundant molecular modules in fission yeast ([Figure 2D][5]). We also determined the overall correlation of our protein data set with mRNA abundance as estimated by cDNA microarray analysis. The comparison of 1367 protein–mRNA pairs revealed a Pearson correlation coefficient ( r P) of 0.58 ([Figure 3A][6]), indicating a substantial correlation between mRNA and protein abundance in fission yeast similar to what was found for budding yeast ([Ghaemmaghami et al , 2003][7]; [Greenbaum et al , 2003][8]). We also calculated correlation coefficients for specific functional pathways, protein families and multisubunit protein complexes. Whereas high coefficients were obtained for signalling and metabolic pathways ([Figure 3B][6]), the majority of multisubunit protein complexes showed very low or even negative correlation coefficients ([Figure 3B][6]). The poor protein–mRNA correlation for complexes would be expected, if their subunits were coordinately regulated. Coordinate regulation may lead to clustering of protein–mRNA ratios for complex components around a similar value, thus precluding a strong correlation. Indeed, we noticed more...