Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine.
Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses. Metabolic labeling allows cells from different experiments to be mixed prior to analysis. This approach has been reported for prokaryotic cells. Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae. Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins. D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000). Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media. Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S. cerevisiae. Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.
Jiang
Heng
H
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada H3G 1M8.
English
Ann M
AM
D015180:Electrophoresis, Gel, Two-Dimensional; D007554:Isotopes / Q000737:chemistry / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007554:Isotopes; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D007930:Leucine