Pushing the Limits of Sensitivity: Micropillar Array-Based Chromatography Coupled to a Quadrupole Orbitrap Mass Spectrometer and FAIMS for Low-Input Proteomics

Importance of the Topic

The ability to perform proteome profiling on extremely small sample inputs addresses key challenges in fields such as single-cell analysis, rare cell population studies and clinical diagnostics. Improving chromatographic sensitivity and reducing chemical noise are essential to uncover low-abundance proteins and deepen our understanding of cellular heterogeneity.

Study Objectives and Overview

This work aims to push the limits of sensitivity in liquid chromatography–mass spectrometry (LC-MS) by combining a novel non-porous micropillar array column (µPAC™) with high-field asymmetric waveform ion mobility spectrometry (FAIMS) and a quadrupole Orbitrap mass spectrometer. The goal is to demonstrate reliable, deep proteome profiling from sub-nanogram amounts of HeLa and K562 cell digests.

Methodology and Instrumentation

The study employed serial dilutions of HeLa and K562 cell lysate digests from 10 ng down to 500 pg, simulating the protein content of a few cells. Peptides were separated on a prototype non-porous µPAC column operated at 50 °C and 250 nL/min over a 60 min gradient. FAIMS Pro was used to filter out singly charged background ions by applying specific compensation voltages, enhancing the transmission of multiply charged peptide ions. Mass analysis was carried out on a Thermo Scientific™ Orbitrap Exploris™ 480.

Instrumentation

• Thermo Scientific Orbitrap Exploris 480 mass spectrometer
• FAIMS Pro interface for ion selection
• Prototype µPAC™ non-porous pillar array column (PharmaFluidics)
• IonOpticks column (25 cm) used for FAIMS optimization

Data Processing

Raw data were searched with Proteome Discoverer™ 2.5 using Sequest with and without multiple precursor search (MPS) and a predicted spectral library generated by Prosit. Results were filtered at 1 % false discovery rate (FDR) at peptide and protein levels.

Main Results and Discussion

• FAIMS filtering markedly reduced chemical noise and improved signal-to-noise ratio for low-abundance peptides.
• The µPAC column delivered sharp peaks with retention time variation below 0.3 % and peak area CVs under 12 % at picogram loads.
• From just 500 pg of HeLa digest, more than 1 500 protein groups were identified; at 1 ng, identifications exceeded 4 000 proteins with consistent technical reproducibility.
• Spectral library searching with Prosit achieved the highest proteome coverage compared to conventional Sequest searches.

Benefits and Practical Applications

By combining µPAC chromatography with FAIMS and high-resolution Orbitrap detection, researchers can:

• Analyze proteomes from single cells or tiny tissue biopsies.
• Improve depth and reproducibility in low-input workflows.
• Enhance sensitivity for biomarker discovery in clinical and pharmaceutical research.

Future Trends and Applications

Advances in microfabricated column designs, optimized ion mobility separations and AI-driven data analysis will further increase proteome coverage from minimal sample amounts. Integration with microfluidic cell isolation, higher-throughput FAIMS devices and improved spectral libraries will drive single-cell proteomics toward routine use in personalized medicine and systems biology.

Conclusion

The synergistic use of a non-porous µPAC pillar array column, FAIMS Pro ion mobility filtering and an Orbitrap Exploris 480 mass spectrometer significantly enhances sensitivity and reproducibility in low-input proteomics. This platform paves the way for routine single-cell analyses and deep proteome profiling of limited material.

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