Quantitative Proteomics of Metabolic Enzymes in S. cerevisiae Using Triple Quadrupole LC/MS/MS

Importance of the Topic

Understanding the abundance of metabolic enzymes in Saccharomyces cerevisiae is essential for optimizing microbial cell factories used in food fermentation, chemical synthesis, pharmaceuticals and biofuels. Precise quantification of these proteins enables insight into pathway regulation and cellular dynamics.

Study Objectives and Overview

This work applies targeted quantitative proteomics using triple quadrupole LC-MS/MS with multiple reaction monitoring to compare enzyme expression in wild-type and single gene deletion strains of S. cerevisiae under stable isotope labeling.

Methodology and Instrumentation

Cultures of wild-type S288C and BY4742 deletion mutants (gnd1Δ, pfk1Δ, zwf1Δ) were grown in media with labeled and unlabeled glucose. Proteins were digested into tryptic peptides and 2 856 MRM transitions were monitored for 303 peptides (137 proteins) in S288C and 409 peptides (199 proteins) in BY4742. Ultra fast mass spectrometry ensured high-speed scanning for complex mixtures.
Instrumentation details:

• HPLC: Prominence Nano
• MS: Shimadzu LCMS-8040 with nano-ESI interface
• Mobile phase A: 0.1% formic acid in water
• Mobile phase B: 0.1% formic acid in acetonitrile
• Flow rate: 400 nL/min
• Gradient: 10% B to 40% B over 45 min
• Column: L-Column ODS, 0.1 mm I.D. × 150 mm
• MRM library: Central metabolic enzymes in yeast

Main Results and Discussion

MRM chromatograms demonstrated clear detection of Gnd1p transitions and confirmed the expected depletion in the gnd1Δ mutant. Dwell-time experiments showed that reliable quantification is maintained at 1 ms dwell and pause times, highlighting UFMS benefits. Comparative profiling across glycolysis, glycerol biosynthesis, TCA cycle and NADPH regeneration revealed differential enzyme levels consistent with genotypes, while zwf1Δ and gnd1Δ exhibited distinct expression patterns despite shared pathway roles.

Benefits and Practical Applications

Key advantages include:

• Antibody-free multiplex analysis
• High sensitivity and specificity for low-abundance targets
• Rapid cycle times for dynamic studies
• Applicability in strain engineering, quality control and metabolic flux investigations

Future Trends and Potential Applications

Advances in ultra fast mass spectrometry promise expanded proteome coverage and deeper insight into dynamic metabolic changes. Integration with flux analysis and real-time monitoring in bioprocesses will drive innovation in microbial engineering. Extension of this approach to other organisms and post-translational modifications is anticipated.

Conclusion

Triple quadrupole LC-MS/MS with multiple reaction monitoring and ultra fast scanning provides a robust, high-throughput platform for targeted quantification of yeast metabolic enzymes, supporting detailed pathway analysis without immunoassays.

References

Matsuda F, Ogura T, Tomita A, Hirano I, Shimizu H. Nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode based quantitative platform for analyzing multiple enzymes associated with central metabolic pathways of Saccharomyces cerevisiae using ultra fast mass spectrometry. J Biosci Bioeng. 2015 Jan;119(1):117–20.