Quantitative targeted nano- and capillary- flow LC-MS peptide analysis using the Vanquish Neo UHPLC System coupled to a triple quadrupole mass spectrometer

Importance of the Topic

The combination of low-flow liquid chromatography with mass spectrometry has been a cornerstone of proteomics for its high sensitivity when analyzing complex peptide mixtures. However, nano-flow methods often face challenges in robustness, throughput, and ease of use for large sample cohorts. Capillary-flow LC-MS offers a promising compromise, delivering high sensitivity alongside greater sample throughput and operational stability.

Study Objectives and Overview

This study evaluated the performance of a Thermo Scientific Vanquish Neo UHPLC system coupled to a TSQ Altis triple quadrupole mass spectrometer for quantitative targeted peptide analysis. By comparing nano-flow (0.3 µL/min) and capillary-flow (3.0 µL/min) workflows on the same hardware, the goal was to assess sensitivity, linearity, throughput, and long-term robustness in HeLa digest samples spiked with heavy-labeled PRTC peptides.

Methodology

Peptide standards (PRTC) were serially diluted in a HeLa protein digest matrix to concentrations from 0.01 to 100 fmol/µL. Nano-flow experiments used a 75 µm × 15 cm EASY-Spray PepMap Neo column with a 27 min gradient, while capillary-flow used a 150 µm × 15 cm column with a 12.4 min gradient. The TSQ Altis operated in selected reaction monitoring mode at 600 SRMs/s, ensuring >12 data points across each peak. Method performance was assessed for precision, accuracy, limit of quantitation (LoQ), peak shape, linearity, and carry-over, following FDA bioanalytical guidance.

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system
• Thermo Scientific TSQ Altis Triple Quadrupole Mass Spectrometer
• Thermo Scientific EASY-Spray PepMap Neo columns (75 µm and 150 µm ID, 2 µm C18)
• Thermo Scientific nanoViper fitting system
• Chromeleon 7.2.10 MUd software for LC control

Key Results and Discussion

• Analysis time was reduced by ~60% in capillary-flow (203 min for 10 runs) versus nano-flow (504 min).
• Linearity for 15 PRTC peptides was excellent (average r = 0.9988 nano, 0.9993 capillary) over 0.01–100 fmol/µL.
• Limits of quantitation were comparable: most peptides achieved 100 amol/µL quantitation in both modes.
• Peak widths at half-height decreased by 75–80% in capillary-flow, enhancing signal-to-noise and resolution.
• Long-term robustness: 200 injections over 75 h showed retention time RSD <0.28% and peak area RSD ~5.3%.
• Carry-over remained low (<0.1%), with system contributions below 0.07%.

Benefits and Practical Applications

The capillary-flow LC-MS workflow on a unified Vanquish Neo platform combines high sensitivity with improved throughput and operational stability. It is particularly well suited for targeted peptide quantitation in clinical research, biomarker validation, QA/QC labs, and large cohort studies.

Future Trends and Applications

Advances may include further miniaturization of flow paths, integration of microfluidic devices, higher multiplexing in SRM/MRM assays, and automated sample handling to further boost throughput. Wider adoption of capillary-flow methods can bridge the gap between discovery proteomics and routine high-throughput screening.

Conclusion

The Vanquish Neo UHPLC system coupled to TSQ Altis delivers robust, reproducible, and highly sensitive targeted peptide quantitation at both nano- and capillary-flow rates. Capillary-flow methods achieve significant time savings without compromising analytical performance, providing a versatile solution for modern proteomic workflows.

References

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