An integrated LC-MS system performance evaluation test for peptide mapping and monitoring

Significance of Topic

Peptide mapping by LC–MS is a cornerstone technique for verifying amino acid sequences and molecular attributes of biotherapeutics throughout development and quality control.

Study Objectives

This work presents an integrated LC–MS system performance evaluation test (SET) that uses a pre-digested BSA standard and automated eWorkflows to assess key chromatographic and mass spectrometric metrics for peptide mapping and monitoring.

Methodology and Instrumentation

• Sample: Thermo Scientific Pierce BSA protein digest standard at 1 pmol/µL.
• Chromatography: Hypersil GOLD VANQUISH™ C18 column (150 × 2.1 mm, 1.9 µm) on Vanquish Flex or Horizon UHPLC at 50 °C, 0.25 mL/min gradient.
• Mass spectrometry: Orbitrap Exploris 240 or Exploris MX at 120 000 resolution over m/z 280–1600; data-dependent MS2 on Exploris 240.
• Software: Chromeleon™ CDS eWorkflow automates sequence setup, acquisition, processing, and pass/fail reporting with defined acceptance criteria.

Main Results and Discussion

• Composite scoring: Achieved mass accuracy ≤ 5 ppm, isotopic dot product ≥ 0.9, and apex alignment ≤ 0.5 across five representative peptides.
• General system check: Retention time RSD < 2 %, area RSD < 10 %, peak width ≤ 10 s, and consistent TIC overlays demonstrated robust reproducibility.
• Special system check: Monitored deamidation levels of HLVDEPQNLIK variants (%CV < 10 %) and retention shifts between modified and unmodified peptides within defined windows.
• Sequence coverage: Automated calculation using 43 tryptic peptides yielded ≥ 80 % coverage across multiple BSA lots, confirming sample integrity.
• MS2 fragmentation: Exploris 240 met y-ion abundance ranges for y2, y3, and y6 fragments, validating collision energy settings.

Practical Benefits and Applications

• Rapid assessment of LC–MS system readiness via a single eWorkflow from sample queue to pass/fail report.
• Compliance-ready solution supports regulated environments by embedding acquisition, processing, and reporting methods.
• Standardized metrics enable long-term system performance monitoring and column screening decisions.

Future Trends and Opportunities

Advances in LC-HRAM-MS will drive deeper proteoform characterization, integration of AI-based data evaluation, and broader adoption of standardized performance tests to enhance throughput, robustness, and regulatory alignment.

Conclusion

The described SET and Chromeleon CDS eWorkflow deliver a streamlined, reproducible framework for qualifying LC–MS systems for peptide mapping and monitoring, ensuring consistent performance in research and quality control labs.

References

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