Sensitive and High-throughput Single-cell Proteomics Workflow on New Quadrupole-ion trap-Orbitrap Mass Spectrometer with FAIMS Separation

Importance of the Topic

The study of protein expression at the single-cell level addresses the limitations of bulk proteomics by revealing cellular heterogeneity and rare cell populations. Advances in sample processing, separation, and detection are vital for profiling minute proteomes without compromising sensitivity or throughput.

Objectives and Study Overview

This work integrates nanoPOTS microdroplet processing, tandem mass tag (TMT) isobaric labeling, and a quadrupole-ion trap-Orbitrap mass spectrometer equipped with FAIMS Pro Interface and real-time search to enhance single-cell proteome coverage and quantitation accuracy. Both label-free and multiplexed TMT workflows were evaluated on cultured HeLa and murine cell types.

Materials and Methods

Single cells from HeLa and murine lines were isolated by fluorescence-activated sorting, lysed, digested, and labeled (for TMT experiments) within nanoPOTS nanowells. Peptides were trapped on a short SPE column and separated on low-flow nanoLC columns (20 µm i.d. for label-free, 30 µm i.d. for TMT) using an UltiMate 3000 RSLCnano system.
Data acquisition employed the Thermo Scientific Orbitrap Eclipse Tribrid MS with FAIMS Pro Interface, alternating compensation voltages to remove singly charged noise. Label-free analysis used high-resolution MS2 in the Orbitrap, while TMT10plex experiments compared MS2 and SPS MS3 with real-time search.

Used Instrumentation

• Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer
• FAIMS Pro Interface
• Thermo Scientific UltiMate 3000 RSLCnano system
• nanoPOTS (Nanodroplet Processing in One‐pot for Trace Samples) platform
• PRSO-V2 Sonation column oven
• Proteome Discoverer 2.4 and MaxQuant software

Main Results and Discussion

• Label-free single HeLa cell analysis with FAIMS yielded on average 829 protein groups, a 3-fold increase over non-FAIMS runs. From 0.5 ng digest, >2 000 protein groups and >8 500 peptides were identified at <1% FDR.
• TMT10plex single-cell analysis of murine epithelial, endothelial, and immune cells achieved clear cell type discrimination. SPS MS3 with real-time search improved quantitative accuracy and differential protein coverage versus MS2 alone, identifying up to 4 781 peptides and 2 346 proteins.
• High-throughput workflows processed 24–40 single cells per study, maintaining reproducible quantitation of ~2 000 protein groups across cells.

Benefits and Practical Applications

• Ultra-sensitive detection down to sub-nanogram protein inputs enables profiling of individual mammalian cells.
• FAIMS Pro Interface reduces chemical noise, boosting identification of low-abundance peptides in label-free workflows.
• TMT multiplexing with SPS MS3 and real-time search enhances quantitation accuracy for differential expression studies.
• NanoPOTS sample handling minimizes losses, supporting robust analysis of rare or precious cell populations.

Future Trends and Applications

• Further increases in throughput using automated nanoPOTS and parallelized mass spectrometry.
• Integration with microfluidic cell sorting and spatial proteomics to map tissue heterogeneity.
• Expansion to clinical specimens such as circulating tumor cells for precision diagnostics.
• Combination with emerging separation technologies (e.g., capillary electrophoresis) for deeper coverage.

Conclusion

This workflow demonstrates that coupling nanoPOTS, FAIMS-enhanced ultralow-flow LC, and advanced Orbitrap detection enables comprehensive, high-throughput single-cell proteomics in both label-free and TMT formats. The approach offers significant gains in sensitivity, coverage, and quantitation accuracy, paving the way for detailed cellular heterogeneity studies.

Reference

1. Zhu Y., et al. Angew. Chemie Int. Ed. 57, 12370 (2018)
2. Zhu Y., et al. Nat. Commun. 9, 882 (2018)
3. Budnik B., et al. Genome Biol. 19, 161 (2018)