Advancing low flow LC/MS for single cell proteomics with variable flow and 50 cm microfabricated pillar array columns

Importance of the Topic

The ability to analyze proteins at the single-cell level uncovers cellular heterogeneity critical for understanding complex biological processes, disease mechanisms, and drug responses. Optimizing chromatographic separation and mass spectrometry workflows for low-input samples enhances sensitivity and throughput, enabling routine single-cell proteomics in research and industrial laboratories.

Aims and Overview of the Study

This study introduces a low-flow LC–MS approach using 50 cm microfabricated pillar array columns (µPAC Neo Plus) coupled to an Orbitrap Exploris 240 mass spectrometer. The goals are to maximize proteome coverage from single HeLa cells, evaluate the impact of variable flow rates on sensitivity and throughput, and benchmark results against bulk HeLa digest dilution series.

Methodology and Instrumentation

HeLa single cells were isolated with the cellenONE platform into 384-well LoBind plates and processed via a one-pot lysis and digestion protocol. Samples were directly aspirated from plate wells into a Vanquish Neo UHPLC system configured in a back-flush trap-and-elute workflow.

• Low-volume sample handling in Eppendorf LoBind 384-well plates
• Chromatography on 50 cm µPAC Neo Plus columns with a 10 µm ID voltage spacer and external heating
• Variable elution flow rates of 100 and 200 nL/min
• DIA acquisition on an Orbitrap Exploris 240 (MS1 resolution 120 k; MS2 resolution up to 120 k)

Main Results and Discussion

Optimizing column connectivity and reducing flow rates significantly improved chromatographic performance and proteome depth. Key findings include:

• At 100 nL/min, over 2000 protein groups were identified from single HeLa cells.
• Flow reduction from 250 nL/min to 100 nL/min yielded a 25–40% increase in proteome coverage.
• Protein abundance variability was higher in single cells versus bulk dilutions, reflecting biological heterogeneity.
• Proteome coverage correlated strongly with cell size, with larger HeLa cells producing deeper profiles.

Benefits and Practical Applications

The presented workflow delivers:

• Exceptional sensitivity for low-input and single-cell analyses.
• Enhanced throughput via trap-and-elute configuration and variable flow methods.
• Automatable sample handling compatible with standard plate formats.

Future Trends and Applications

Future developments may focus on integrating microfluidic enrichment, further miniaturizing chromatography, and exploiting artificial intelligence for data processing. Multiplexed single-cell proteomics and real-time acquisition strategies are poised to enhance clinical diagnostic and drug discovery applications.

Conclusion

This work demonstrates that low-flow LC–MS with 50 cm pillar array columns and optimized connectivity substantially enhances single-cell proteome coverage and workflow efficiency, establishing a robust platform for high-sensitivity proteomics.

Reference

1. Matzinger M, Müller E, Dürnberger G, Pichler P, Mechtler K. Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics. Anal Chem. 2023;95(9):4435–4445.