MaxPeak High Performance Surfaces Enable Reproducibility and Enhanced Experimental Robustness in High Throughput Proteomics

Significance of the Topic

Large-scale clinical proteomics demands LC-MS methods that ensure consistent performance over extended periods and numerous samples. Robust chromatographic separation and stable mass spectrometric detection are critical to generate reliable quantitative and qualitative data for biomarker discovery and cohort studies.

Objectives and Study Overview

This study evaluated the reproducibility and robustness of a MaxPeak High Performance Surfaces-treated LC system (ACQUITY PREMIER) coupled to a SYNAPT XS mass spectrometer. A complex E. coli tryptic digest served as a QC sample, injected over 180 times across a six-week, high-throughput proteomic experiment, to assess key analytical metrics.

Methodology

• QC injections of 2 µg E. coli digest every four hours between clinical plasma digests over six weeks (≈180 injections).
• Chromatography: 2.1 mm×100 mm UPLC CSH column at 55 °C; flow rate 150 µL/min; 1–35% acetonitrile gradient in 16 min; sample tray at 8 °C.
• Mass spectrometry: SYNAPT XS in positive electrospray mode; ToF HDMSE acquisition (50–2000 Da); transfer collision energy ramp.

Used Instrumentation

• LC system: ACQUITY PREMIER with MaxPeak HPS surfaces
• Column: ACQUITY UPLC CSH 2.1 mm × 100 mm (HPS)
• Mass spectrometer: SYNAPT XS (Waters)
• Software: MassLynx v4.2, Progenesis QI for Proteomics, PLGS 3.0.3, Spotfire

Main Results and Discussion

• Chromatographic reproducibility: retention time CV ≤1% across injections; peak widths mainly ~3 s FWHM.
• Peptide intensity stability: consistent summed product‐ion intensities for both high‐ and low‐abundance proteins throughout the study.
• Mass accuracy: >95% of peptide precursors measured within ±5 ppm of theoretical mass.
• Protein sequence coverage: average 76% coverage for a model protein (CV 2.8%); overall coverage distribution peaked at 40–49% for all proteins.
• Protein identifications: ~76% of proteins consistently identified in early, mid, and late QC injections, demonstrating sustained detection capability.

Benefits and Practical Applications

• Minimized adsorption of analytes to metal surfaces, enhancing detection sensitivity.
• Reliable, high‐throughput analytical‐scale LC-MS workflows suitable for large clinical cohorts.
• Reduced technical variability supports confident biomarker discovery and longitudinal studies.

Future Trends and Potential Applications

Integration of MaxPeak HPS-based systems with advanced acquisition strategies (e.g., data-independent acquisition and ion mobility) may further enhance depth of coverage and data quality. Automation, AI-driven QC monitoring, and expansion to diverse sample types will drive broader adoption in clinical and translational proteomics.

Conclusion

This evaluation demonstrates that MaxPeak High Performance Surfaces on the ACQUITY PREMIER, combined with SYNAPT XS, deliver exceptional robustness and reproducibility over extended, high-throughput proteomic analyses, meeting the rigorous demands of large‐scale clinical studies.

References

• Lennon S., Hughes C. J., Muazzam A., Townsend P. A., Gethings L. A., Wilson I. D., Plumb R. S. J. Proteome Res. DOI:10.1021/acs.jproteome.0c00821.
• DeLano M., Walter T. H., Lauber M. A., Gilar M., Jung M. C., Nguyen J. M., Boissel C., Patel A. V., Bates-Harrison A., Wyndham K. D. Anal. Chem. 2021, 93, 5773–5781.
• Distler U., Kuharev J., Navarro P., Levin Y. Nat. Methods 2014, 11, 167–170.