Long-term stability and reproducibility of nano, capillary and micro-flow LC-MS separations

Importance of the Topic

The performance of nano and micro-flow LC-MS is crucial for high-sensitivity proteomics, enabling detailed biomarker discovery and quantitative studies. Long-term reproducibility and system robustness underpin reliable data across extended campaigns and laboratories.

Objectives and Study Overview

This work evaluates the six-month stability and intersystem consistency of Vanquish Neo UHPLC platforms with PepMap Neo columns, analyzing nano, capillary, and micro-flow separations coupled to Orbitrap Exploris and TSQ Altis instruments across multiple sites.

Methodology and Instrumentation

• UHPLC: Vanquish Neo with temperature-controlled column compartment.
• Columns: PepMap Neo (75 µm × 50 cm) for nano and Acclaim PepMap for micro-flow.
• Mass spectrometry: Orbitrap Exploris 240/480 (DDA) and TSQ Altis (MRM).
• Mobile phases: 0.1% formic acid in water (A) and 0.1% formic acid in 80% acetonitrile (B).
• Data analysis: Proteome Discoverer 2.5 (Sequest HT, INFERYS) and Chromeleon 7.2.10 for LC-UV metrics.
• Standards: BSA, Cytochrome C, and HeLa digest with PRTC peptides.

Key Results and Discussion

• BSA nanoLC over 1,600 injections showed consistent peak widths and backpressure fluctuations <25 bar over six months.
• Cytochrome C retention times remained within 0.3 min SD and <0.2% RSD across six UHPLC systems.
• HeLa digest profiling identified ~33,000 peptides and 4,400 proteins with 4.1% peptide and 2.2% protein group variation.
• Micro-flow method (14.4 min cycle) maintained <3 bar pressure variation and <0.5% RT RSD for 12 PRTC peptides over 760 injections.

Benefits and Practical Applications

High stability and reproducibility in low-flow separations support label-free quantitation, interlaboratory standardization, and high-throughput workflows, benefiting proteomics research, clinical assays, and quality control in industrial settings.

Future Trends and Opportunities

• Automated sample handling integrated with low-flow LC systems to boost throughput and consistency.
• Real-time system diagnostics and predictive maintenance for uninterrupted operation.
• Innovations in column chemistries and UHPLC hardware targeting sub-0.1% RT variability.
• Enhanced data analysis pipelines combining robust chromatography with machine-learning algorithms for improved quantitation.

Conclusion

Vanquish Neo UHPLC and PepMap Neo columns deliver exceptional long-term robustness and reproducibility for nano and micro-flow proteomics. Consistent chromatographic performance across systems and extended operation demonstrates their suitability for demanding large-scale proteomic applications.

Reference

1. Bian Y., Zheng R., et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS. Nat Commun. 2020;11:157.
2. Bian Y., Bayer F. P., et al. Robust Microflow LC-MS/MS for Proteome Analysis: 38,000 Runs and Counting. Anal Chem. 2021;93(8):3686–3690.