Targeted Proteomic Analysis of Central Metabolic Enzymes Using Nano-LC-Triple Quadrupole Mass Spectrometry

Importance of the topic

Targeted proteomics using nano-LC combined with triple quadrupole mass spectrometry enables precise quantification of central metabolic enzymes even in samples with low peptide abundance. This approach is crucial in metabolic engineering to optimize biosynthesis of valuable compounds and in medical research to understand disease-related metabolic reprogramming, such as the Warburg effect in cancer cells.

Objectives and Study Overview

This application study aimed to evaluate the stability, speed, and transferability of a targeted proteomic platform for analyzing central metabolic enzymes in yeast (Saccharomyces cerevisiae, Lipomyces starkeyi) and human MCF-7 cells. Key objectives included assessing nano-LC retention time reproducibility, high-speed data acquisition performance, cross-platform MRM method transfer, and the impact of single-gene knockouts on enzyme expression profiles.

Methodology and Instrumentation

• Sample preparation: cell culture, protein extraction, reductive alkylation, trypsin digestion, desalination using MonoSpin C18 or StageTips.
• Chromatography: nano-LC with LC-20ADnano, low-flow gradient (400 nL/min) on L-column Micro C18 columns.
• Mass spectrometry: triple quadrupole MS with LCMS-8040 and LCMS-8060 UFMS technology, operated in MRM mode.
• Data analysis: Skyline software, custom MRM libraries for yeast and human targets, collision energy calculation (CE = 0.03 × Q1 m/z + 4.0).
• Quality control: weekly ion source cleaning and retention time markers (FMR-002) to ensure retention stability.

Main Results and Discussion

• Retention time stability: nano-LC retention shifts remained within 0.2 min over one month, confirming robustness for long-term studies.
• High-speed acquisition: LCMS-8060 achieved over 500 MRM channels/sec with dwell times as low as 1 ms, without compromising quantitative accuracy (relative variation < 2-fold).
• Method transfer: published human MCF-7 MRM assay (133 peptides, 398 transitions) was successfully transferred between Thermo Scientific and Shimadzu platforms, retaining retention time correlation.
• Knockout analysis in S. cerevisiae:
  • pdc1Δ strain: absence of Pdc1, compensatory upregulation of Pdc5, Hxk1, Tdh1 indicating pathway adaptation.
  • gcr2Δ strain: global downregulation of glycolytic enzymes, altered amino acid biosynthesis and trehalose pathway, demonstrating wide metabolic impact of a single regulator.

Benefits and Practical Applications of the Method

• Allows simultaneous quantification of dozens to hundreds of enzymes with high sensitivity.
• Facilitates metabolic engineering by mapping enzyme expression changes in engineered strains.
• Provides insights into disease-related metabolic shifts for biomarker discovery.
• Supports method transferability and reproducibility across laboratories and instrument platforms.

Future Trends and Opportunities

Directed expansion of MRM assays toward genome-wide coverage will enable comprehensive metabolic profiling in diverse organisms. Advances in instrument speed, sensitivity, and data analysis pipelines will further enhance throughput. Integration with CRISPR-based editing and single-cell proteomics may open new frontiers in metabolic regulation and precision medicine.

Conclusion

The integration of nano-LC and ultra-fast triple quadrupole mass spectrometry constitutes a reliable and high-throughput targeted proteomics platform for quantitative analysis of central metabolism. This approach demonstrates stable performance, rapid data acquisition, and method transferability, offering valuable tools for both industrial biotechnology and biomedical research.

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