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. 1999 Mar;19(3):1720-30.
doi: 10.1128/MCB.19.3.1720.

Correlation between protein and mRNA abundance in yeast

S P Gygi  1 , Y Rochon, B R Franza, R Aebersold
Affiliations

Correlation between protein and mRNA abundance in yeast

S P Gygi et al. Mol Cell Biol. 1999 Mar.

Abstract

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243-251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

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Figures

FIG. 1
FIG. 1
Schematic illustration of proteome analysis by 2DE and mass spectrometry. In part I, proteins are separated by 2DE, stained spots are excised and subjected to in-gel digestion with trypsin, and the resulting peptides are separated by on-line capillary high-performance liquid chromatography. In part II, a peptide is shown eluting from the column in part I. The peptide is ionized by electrospray ionization and enters the mass spectrometer. The mass of the ionized peptide is detected, and the first quadrupole mass filter allows only the specific mass-to-charge ratio of the selected peptide ion to pass into the collision cell. In the collision cell, the energized, ionized peptides collide with neutral argon gas molecules. Fragmentation of the peptide is essentially random but occurs mainly at the peptide bonds, resulting in smaller peptides of differing lengths (masses). These peptide fragments are detected as a tandem mass (MS/MS) spectrum in the third quadrupole mass filter where two ion series are recorded simultaneously, one each from sequencing inward from the N and C termini of the peptide, respectively. In part III, the MS/MS spectrum from the selected, ionized peptide is compared to predicted tandem mass spectra computer generated from a sequence database. Provided that the peptide sequence exists in the database, the peptide and, by association, the protein from which the peptide was derived can be identified. Unambiguous protein identification is attained in a single analysis because multiple peptides are identified as being derived from the same protein.
FIG. 2
FIG. 2
2D silver-stained gel of the proteins in yeast total cell lysate. Proteins were separated in the first dimension (horizontal) by isoelectric focusing and then in the second dimension (vertical) by molecular weight sieving. Protein spots (156) were chosen to include the entire range of molecular weights, isoelectric focusing points, and staining intensities. Spots were excised, and the corresponding protein was identified by mass spectrometry and database searching. The spots are labeled on the gel and correspond to the data presented in Table 1. Molecular weights are given in thousands.
FIG. 3
FIG. 3
Tandem mass (MS/MS) spectra resulting from analysis of a single spot on a 2D gel. The first quadrupole selected a single mass-to-charge ratio (m/z) of 687.2 (A) or 592.6 (B), while the collision cell was filled with argon gas, and a voltage which caused the peptide to undergo fragmentation by CID was applied. The third quadrupole scanned the mass range from 50 to 1,400 m/z. The computer program Sequest (8) was utilized to match MS/MS spectra to amino acid sequence by database searching. Both spectra matched peptides from the same protein, S57593 (yeast hypothetical protein YMR226C). Five other peptides from the same analysis were matched to the same protein.
FIG. 3
FIG. 3
Tandem mass (MS/MS) spectra resulting from analysis of a single spot on a 2D gel. The first quadrupole selected a single mass-to-charge ratio (m/z) of 687.2 (A) or 592.6 (B), while the collision cell was filled with argon gas, and a voltage which caused the peptide to undergo fragmentation by CID was applied. The third quadrupole scanned the mass range from 50 to 1,400 m/z. The computer program Sequest (8) was utilized to match MS/MS spectra to amino acid sequence by database searching. Both spectra matched peptides from the same protein, S57593 (yeast hypothetical protein YMR226C). Five other peptides from the same analysis were matched to the same protein.
FIG. 4
FIG. 4
Current proteome analysis technology utilizing 2DE without preenrichment samples mainly highly expressed and long-lived proteins. Genes encoding highly expressed proteins generally have large codon bias values. (A) Distribution of the yeast genome (more than 6,000 genes) based on codon bias. The interval with the largest frequency of genes is 0.0 to 0.1, with more than 2,500 genes. (B) Distribution of the genes from identified proteins in this study based on codon bias. No genes with codon bias values less than 0.1 were detected in this study. (C) Distribution of identified proteins in this study based on predicted half-life (estimated by N-end rule).
FIG. 5
FIG. 5
Correlation between protein and mRNA levels for 106 genes in yeast growing at log phase with glucose as a carbon source. mRNA and protein levels were calculated as described in Materials and Methods. The data represent a population of genes with protein expression levels visible by silver staining on a 2D gel chosen to include the entire range of molecular weights, isoelectric focusing points, and staining intensities. The inset shows the low-end portion of the main figure. It contains 69% of the original data set. The Pearson product moment correlation for the entire data set was 0.935. The correlation for the inset containing 73 proteins (69%) was only 0.356.
FIG. 6
FIG. 6
Effect of highly abundant proteins on Pearson product moment correlation coefficient for mRNA and protein abundance in yeast. The set of 106 genes was ranked according to protein abundance, and the correlation value was calculated by including the 40 lowest-abundance genes and then progressively including the remaining 66 genes in order of abundance. The correlation value climbs as the final 11 highly abundant proteins are included.
FIG. 7
FIG. 7
Relationship between codon bias and protein and mRNA levels in this study. Yeast mRNA and protein expression levels were calculated as described in Materials and Methods. The data represent the same 106 genes as in Fig. 5.
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