LC-MS: Ultra-robust micro-flow LC-MS/MS for targeted high-throughput peptide quantification using the Vanquish Neo UHPLC system

Significance of the Topic

Increasing throughput and robustness in peptide quantification is critical for large scale proteomic validation studies where nano flow LC MS methods face limitations in sample loading and reproducibility.

Objectives and Study Overview

This work adapts a previously published high throughput micro flow LC MS MS approach to the new Vanquish Neo UHPLC platform aiming at a robust method capable of quantifying standard peptides at 100 samples per day with consistent performance.

Methodology

HeLa protein digest spiked with heavy labeled PRTC peptides was prepared at 50 ng per microliter and 25 fmol peptide concentration. Samples were analyzed in direct injection micro flow mode with a 1.0 mm ID x 15 cm column, operating 100 microliters per minute for loading and washing and 50 microliters per minute during the gradient phase. Gradient conditions delivered 1 percent to 35 percent organic over 8.5 minutes with an overall cycle time of 14.4 minutes. Selected reaction monitoring transitions were monitored for twelve PRTC peptides. A sequence of 760 injections over 7.5 days assessed retention time, pressure and peak area stability.

Used Instrumentation

• Vanquish Neo UHPLC system with Binary Pump N and Split Sampler NT
• Acclaim PepMap 1.0 mm ID x 15 cm column
• Thermo Scientific TSQ Altis triple quadrupole mass spectrometer with OptaMax NG HESI source
• 50 micrometer ID nanoViper capillaries for reduced post column volume
• Chromeleon 7.2.10 or Xcalibur with SII software for data acquisition

Main Results and Discussion

System pressure traces remained superimposable over the entire sequence with less than 3 bar variation. Retention time relative standard deviations were below 0.5 percent for all monitored peptides. Peak area reproducibility was below 10 percent RSD for ten peptides and below 15 percent for the lowest abundant targets. No performance decline was observed over 760 injections.

Benefits and Practical Applications

This micro flow LC MS MS protocol delivers high throughput, excellent reproducibility and robust operation suitable for large sample cohorts in translational proteomics and bioanalytical validation workflows.

Future Trends and Potential Applications

Further integration with automation and sample multiplexing could push throughput beyond 100 samples per day. Advances in column technology and mass spectrometer detection may enable deeper proteome coverage at micro flow rates and shorter cycle times.

Conclusion

The adapted micro flow method on the Vanquish Neo UHPLC system provides a robust, reproducible and high throughput solution for targeted peptide quantification in large scale studies.

Reference

• Boychenko A et al, Technical Note 73208 Tailored high throughput low flow LC MS methods, Thermo Scientific 2019
• Meding S, Boychenko A, Chromatography Today 2016 3 43-45
• Bian Y et al, Robust reproducible quantitative proteomics by micro flow LC MS MS, Nat Commun 2020 11 157
• Bian Y, Bayer FP et al, Robust Microflow LC MS MS for Proteome Analysis 38000 Runs Anal Chem 2021 93 8 3686-3690
• European Medicines Agency Guideline on bioanalytical method validation EMEA CHMP EWP 192217 2009