Efficient tandem capillary flow LC-MS with short μPAC columns and a single ionization source

Significance of the topic

In bottom-up proteomics, increasing sample throughput and maintaining performance is critical. Capillary flow LC-MS offers robust separation at flow rates above 1 μL/min. Tandem LC setup with rapid switching reduces downtime, improving productivity in large-scale proteomic workflows.

Objectives and study overview

• Evaluate productivity, performance, and robustness of a tandem capillary flow LC-MS system using μPAC Neo HT Plus columns in a Vanquish Neo UHPLC platform.
• Optimize three throughput methods: 200 samples per day (SPD), 180 SPD, and 100 SPD, for HeLa cell digest analysis.
• Assess column-to-column reproducibility across production batches.

Instrumentation used

• Thermo Scientific Vanquish Neo UHPLC with Tandem Direct Injection and dual low-dispersion 6-port valves.
• Two μPAC Neo High-Throughput Plus capillary columns configured bidirectionally.
• Thermo Scientific EASY-Spray source with a single integrated liquid junction ESI emitter (20 μMD ID nanoViper line).
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer operated in DIA mode.

Methodology

• HeLa digests prepared at 200 ng/μL in 0.1% TFA/1% ACN, sonicated, diluted, and vortexed.
• 10 μL injection loop minimized overhead time to approximately 1 minute.
• Flow rates of 2.5 μL/min and 1 μL/min were compared; 2.5 μL/min provided sharper peaks and higher identifications.
• DIA settings: MS1 resolution 60k, MS2 resolution 15k, AGC targets 300%/800%, isolation windows 8–15 Th scaled to throughput.
• Data analysis in Spectronaut 19 with 1% FDR threshold; PRTC peptides used for retention time reproducibility assessment.

Key results and discussion

• The tandem workflow increased productivity by 16–33% over direct injection and by 9–17% vs. trap-and-elute workflows.
• At 200 SPD and 2.5 μL/min, ~3 663 protein groups were identified per 200 ng HeLa digest (CV 5–7%).
• Optimized 180 SPD and 100 SPD methods yielded up to 4 713 and 5 692 protein groups, respectively, with median CVs ≤7%.
• Emitter ID selection (15 μMD fused silica vs. 30 μMD steel) had minimal impact on identifications, peak width, and TIC.
• Column reproducibility across 12 columns (4 production batches) was high: retention time CV 1.4–3.3%, protein ID variation ~1.4%.

Benefits and practical applications

• Enhanced throughput supports large-scale proteomic studies and QA/QC analyses.
• Dual-column, single-emitter design reduces downtime without sacrificing chromatographic performance.
• Bidirectional μPAC columns and simplified plumbing eliminate extra grounding requirements.
• Robust retention time stability and low CV facilitate reproducible quantitative workflows.

Future trends and opportunities

• Further reduction of gradient times and adoption of higher-capacity columns to exceed 200 SPD.
• Application in clinical proteomics, biomarker screening, and high-throughput pharmacoproteomics.
• Integration of advanced column chemistries and microfluidic interfaces to minimize dead volume.
• Combination with targeted DIA or PRM workflows for high-throughput quantitative assays.

Conclusion

The tandem capillary flow LC-MS workflow combining μPAC Neo HT Plus columns with the Vanquish Neo UHPLC and a single ESI emitter delivers over 30% productivity gains while maintaining deep proteome coverage and high reproducibility, offering a versatile solution for both discovery and routine proteomics.

References

1. Thermo Fisher Scientific TN003314. A dual-column, single-spray configuration for capillary and micro-flow LC-MS applications.