A dual-column, single-spray configuration for capillary and micro-flow LC-MS applications

Importance of the Topic

Proteomics analyses by LC–MS require high sensitivity, robustness and throughput. Tandem direct injection workflows combining two separation columns with a single electrospray emitter optimize mass spectrometer utilization approaching 100% of cycle time. This enhances robustness by avoiding emitter clogging, increases sample throughput, and maintains quantitative precision across capillary and micro-flow regimes.

Objectives and Study Overview

This study demonstrates the configuration of the Thermo Scientific Vanquish Neo UHPLC system in a tandem direct injection arrangement for capillary (150 µm I.D.) and micro-flow (300–1000 µm I.D.) separations. The aim is to achieve high-throughput bottom-up proteomics with near 100% MS utilization, low carryover, and reproducible quantification.

Methodology and Instrumentation

The workflow employs two columns connected via two low-dispersion switching valves and a single electrospray ionization emitter (Nanospray Flex, EASY-Spray or OptaMax NG) coupled to an Orbitrap Exploris 240 mass spectrometer. Fluidics use nanoViper Fingertight fittings (20 µm for capillary, 50 µm for micro-flow) and PepMap Neo columns (150 µm×150 mm, 300 µm×15 cm, 1000 µm×5 cm/15 cm). Gradient delivery is driven by two Binary Pump N modules—one for separation and one for reconditioning. Data acquisition utilizes data-independent acquisition (DIA) processed with CHIMERYS DIA or Spectronaut 19 software at FDR < 1%.

Main Results and Discussion

• Capillary flow (1–5 µL/min) methods delivered 48–225 samples/day (SPD) with ≥ 90% MS utilization. A 150 µm×150 mm column at 1.5–3.8 µL/min identified 3 111–4 281 protein groups from 50–1000 ng HeLa digest, achieving < 20% CV for over 3500 proteins at 200 ng.
• Micro-flow (15 µL/min, 300 µm×15 cm) achieved 240 SPD and ~ 2 200 proteins at 200 ng with 90% MS utilization; injection of 2000 ng increased identifications to > 3400.
• Milliflow (100 µL/min, 1000 µm×5 cm) reached 277 SPD with 95% MS utilization, yielding ~ 2400 proteins from 1 µg digest.
• Retention time variation across eight columns was < 4%, protein abundance CV < 6%, and carryover remained low (< 0.03% at 180 SPD).
• An inline 75 µm×2 cm trap column extended column lifetime with minimal impact on proteome depth.

Benefits and Practical Applications of the Method

• Easy configuration with standardized fluidic connections.
• Intelligent method creation and valve timing ensure robust, reproducible operation.
• Single-emitter design extends emitter life and eliminates inter-emitter variability.
• High MS utilization maximizes instrument efficiency for high-sensitivity proteomics.
• Low carryover and robust reproducibility suit large-scale studies, QA/QC and industrial analytics.

Future Trends and Potential Applications

• Integration of trap-and-elute schemes for online desalting and further throughput gains.
• Miniaturization of flow regimes for ultra-high sensitivity analyses.
• Automated and AI-driven method optimization to tailor workflows to diverse sample types.
• Extension to other omics fields such as metabolomics and lipidomics.

Conclusion

The tandem direct injection workflow on the Vanquish Neo UHPLC system offers a versatile, high-throughput solution for capillary and micro-flow LC–MS proteomics. It achieves near 100% MS utilization, robust performance, low carryover, and excellent reproducibility, meeting the demands of modern proteomics and industrial applications.

Reference

1. Zheng R, et al. Maximizing sample throughput and sensitivities in nano and capillary LC–MS by leveraging a tandem direct injection workflow and a double-barrel ion source. Thermo Fisher Scientific Technical Note 003335; 2024.
2. Bian Y, et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS. Nature Communications 2020;11:157.
3. Thermo Fisher Scientific. Vanquish Neo System VN-S10 and modules user guide Revision 2.0; 2024.
4. Staes A, et al. High-throughput nanoflow proteomics using a dual-column electrospray source. Analytical Chemistry 2024;96(17):6534–6539.
5. Zheng R, et al. Fast, sensitive, and reproducible nano- and capillary-flow LC–MS methods for high-throughput proteome profiling using the Vanquish Neo UHPLC and Orbitrap Exploris 480 MS. Technical Note 000138; 2021.
6. Meding S, et al. Ultra-robust micro-flow LC–MS/MS for targeted high-throughput peptide quantification using the Vanquish Neo UHPLC system. Technical Note 74161; 2021.