Advancing nanoLC-MS sensitivity for single cell proteomics using solid silicon micro-pillar array column technology

Importance of the Topic

The ability to analyse proteomes from sub-nanogram samples is transforming biological and clinical research by enabling studies at single-cell level or limited material. Optimizing chromatographic performance and ionization efficiency at ultra-low flow rates is key to maximizing sensitivity and throughput in nanoLC-MS proteomics.

Study Objectives and Overview

This work evaluates a novel micro-pillar array column (Thermo Scientific™ 50 cm µPAC™ Neo low-load HPLC column) combined with flow-rate ramping strategies on a standard nanoLC-MS platform. Goals include:

• Increasing proteome coverage from sub-nanogram HeLa protein digest samples.
• Reducing chromatographic overhead times at different sample throughput rates (20–100 samples per day).
• Comparing constant flow and dynamic flow-rate ramping at 250, 125 and 65 nL/min elution rates.

Methodology and Instruments Used

Sample preparation involved Thermo Scientific™ Pierce™ HeLa Protein Digest Standard (50 ng/µL). A Thermo Scientific™ Vanquish™ Neo UHPLC system with a 50 cm µPAC Neo column (75 µm ID) at 40 °C was coupled to an Orbitrap™ Exploris™ 240 mass spectrometer via a NanoViper™ to EASY-Spray™ emitter (10 µm ID). Key LC-MS settings:

• Flow-rate ramping from high initial flow (up to 750 nL/min) down to target eluting flow (250, 125 or 65 nL/min).
• Data-dependent acquisition (Top10) at MS1 120 000 and MS2 60 000 resolution, HCD collision energy 30.
• Proteome Discoverer 3.0 processing using Sequest HT, INFERYS rescoring and CHIMERYS intelligent search (1% FDR).
• Label-free quantification via consensus LFQ workflow; chromatographic metrics with IMP-apQuant.

Main Results and Discussion

Flow-rate ramping reduced column void times from 12–24 min to ~2 min at 125 and 65 nL/min while maintaining linear gradient formation. Key findings include:

• At 250 nL/min, 2862 protein groups were consistently identified from 1 ng HeLa digest; up to 16 % higher coverage was achieved at 65 nL/min versus 250 nL/min.
• Sub-nanogram performance: ~2000 protein IDs from 500 pg and ~1600 from 250 pg digest using optimized ramping at 65 nL/min.
• CHIMERYS search delivered up to three-fold more identifications at 100 SPD compared to Sequest HT alone; for 20 SPD method yields ~2500 IDs.
• Quantification reproducibility: 60–80 % of proteins quantified with CV ≤ 20 % across throughput rates.

Benefits and Practical Applications

This approach offers:

• Enhanced sensitivity for limited-sample and single-cell proteomics.
• Flexible sample throughput (20–100 SPD) without sacrificing depth of coverage.
• Reduced analysis time via fast flow-rate transitions and long µPAC columns operating at moderate pressures.
• Improved quantitative precision through stable low-flow elution.

Future Trends and Applications

Emerging directions include integration of non-porous pillar array columns in automated single-cell platforms, real-time adaptive flow control, and AI-driven data acquisition strategies to further boost sensitivity, throughput and depth in proteomic workflows.

Conclusion

The combination of µPAC Neo 50 cm columns and dynamic flow-rate ramping on conventional nanoLC-MS systems enables robust, high-throughput sub-nanogram proteomics. This method delivers consistent identification of over 2000 proteins from minimal digest amounts with excellent quantitative precision.

Reference

• Op de Beeck J. et al. Advancing nanoLC-MS sensitivity for single cell proteomics using solid silicon micro-pillar array column technology. Thermo Fisher Scientific Poster Notes, 2023.