Ultra-sensitive LC-MS workflow for in-depth label-free analysis of single mammalian cells with nanodroplet sample processing

Importance of the Topic

In-depth proteomic profiling at the single-cell level is crucial for understanding cellular heterogeneity beyond transcriptomics. Label-free mass spectrometry workflows that achieve high sensitivity and broad proteome coverage can reveal functional states, support biomarker discovery, and advance quality control in research and clinical settings.

Study Objectives and Overview

This work aims to integrate ultra-low volume sample processing, high-resolution liquid chromatography separations, and advanced mass spectrometry to enable label-free analysis of individual mammalian cells. By refining key steps—nano-scale digestion, separation, ion mobility filtering, and data analysis—the authors target identification of over 1000 proteins per cell.

Materials and Methods

Sample Processing

• Individual HeLa cells were isolated via inverted microscopy and aspirated into nanoPOTS nanowells (200 nL reaction volume), minimizing surface contact and sample loss.
• Sequential surfactant cleavage, reduction, alkylation, Lys-C digestion, and trypsin digestion were performed in the single nanodroplet.
• Final peptide extracts were transferred directly to an agarose-coated capillary trap column for low-volume introduction.

Chromatographic Separation

• Peptides were separated using a 20 µm i.d. analytical column at ultra-low nanoflow rates coupled to a Thermo Scientific UltiMate 3000 RSLCnano system.

Data Acquisition and Analysis

• A Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer with FAIMS Pro interface was employed to reduce chemical noise and improve signal-to-noise ratios.
• HCD fragmentation and Orbitrap detection provided high-quality MS/MS data.
• Proteome Discoverer 2.4 with SEQUEST and Percolator was used for a two-step search (tryptic and semi-tryptic) at 1% FDR, followed by MaxQuant match-between-runs.

Used Instrumentation

• nanoPOTS nanodroplet processing platform
• Thermo Scientific UltiMate 3000 RSLCnano system
• 20 µm i.d. trap and analytical columns
• Thermo Scientific Orbitrap Eclipse Tribrid MS
• FAIMS Pro interface

Main Results and Discussion

Without FAIMS, analysis of single HeLa cells yielded approximately 400 proteins by MS/MS and 1000 with match-between-runs. Incorporation of FAIMS Pro filtered out singly charged background ions, boosting proteome coverage to around 830 proteins (MS/MS) and 1300 proteins (MS/MS + MBR) per cell. Optimization with 0.5 ng HeLa digest under a two-hour gradient and dual compensation voltages identified over 2000 protein groups and 8500 peptide groups. Compared to the initial workflow (210 proteins per cell), the optimized method achieves nearly fourfold improvement.

Benefits and Practical Applications

• High sensitivity enables label-free profiling of single cells for biomarker discovery and cellular heterogeneity studies.
• Minimal sample loss through nanoPOTS supports analysis of scarce clinical or environmental samples.
• FAIMS Pro reduces chemical noise, improving detection of low-abundance peptides.
• Workflow is compatible with standard proteomics software, facilitating integration in routine QA/QC laboratories.

Future Trends and Applications

Further developments may include automated nanoPOTS handling for higher throughput, integration of additional ion mobility separations, and coupling with emerging data-independent acquisition strategies. Expansion to diverse cell types and multiplexed quantitation approaches will broaden applications in systems biology and precision medicine.

Conclusion

The combination of nanodroplet processing, ultra-low-flow LC separations, FAIMS-enabled Orbitrap Eclipse MS, and refined data analysis establishes a robust label-free workflow for deep proteome profiling of individual mammalian cells. This platform delivers unprecedented sensitivity and coverage, paving the way for routine single-cell proteomics in research and industry.

References

1. Zhu Y., et al. Nat. Commun. 9, 882 (2018)
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3. Schweppe D.K., et al. Anal. Chem. 91(6):4010-4016 (2019)