High-throughput single-cell proteomics analysis with nanodroplet sample processing, multiplex TMT labeling, and ultra-sensitive LC-MS

Significance of Single-Cell Proteomics

Single-cell proteomics offers a direct window into cellular heterogeneity that underlies development, disease progression and treatment response. By identifying and quantifying thousands of proteins at the individual cell level, researchers can reveal rare phenotypes and dynamic processes obscured in bulk analyses. Overcoming the challenges of low sample input, high surface losses and limited throughput is critical to make single-cell proteomics a routine tool in biomedical research and clinical diagnostics.

Objectives and Study Overview

This application note presents a workflow combining nanodroplet processing in one pot for trace samples with multiplexed tandem mass tag labeling to improve throughput and quantitative accuracy in single-cell analysis. The study evaluates MS2 and SPS MS3 strategies enhanced by real-time database searching on an Orbitrap Eclipse Tribrid mass spectrometer to maximize proteome coverage and classification of three cultured murine cell types (epithelial, endothelial and immune cells).

Methodology and Instrumentation

Cells were isolated by fluorescence activated cell sorting into nanoliter wells on a nanoPOTS chip to minimize sample loss. Each cell and a carrier boost sample were subjected to in-well lysis, protein reduction, alkylation, tryptic digestion and TMT10plex labeling in volumes below 200 nanoliters. Labeled peptides were separated by nanoflow liquid chromatography and analyzed on an Orbitrap Eclipse Tribrid system using both MS2 and synchronous precursor selection MS3 with real-time search.

• Liquid chromatography system Thermo Fisher Scientific UltiMate 3000 RSLCnano with 30 micrometer inner diameter 30 centimeter C18 column and integrated electrospray emitter
• Mass spectrometry Orbitrap Eclipse Tribrid with real-time database search and SPS MS3 acquisition
• Data analysis Thermo Fisher Scientific Proteome Discoverer 2.4

Main Results and Discussion

The optimized workflow enabled identification of 2346 protein groups and quantification of 1300 proteins across 40 single cells. Real-time search triggered SPS MS3 doubled identification rates compared to MS2 and improved quantitative accuracy of differentially expressed proteins between cell types. Classification by principal component analysis and heat mapping demonstrated clear separation of epithelial, endothelial and immune cells, validating the robustness of the approach for high-throughput cell typing.

Benefits and Practical Applications

This multiplexed single-cell proteomics strategy delivers:

• High throughput analysis with up to 10 cells per run using TMT barcoding
• Deep proteome coverage of over 2000 proteins per cell
• Enhanced quantification accuracy and reproducibility
• Minimal sample loss by nanodroplet processing

Future Trends and Applications

Advances in sample processing, mass spectrometer speed and data analysis will drive further improvements in sensitivity and throughput. Potential applications include:

• Proteomic profiling of rare clinical specimens such as circulating tumor cells
• Integration with spatial omics for tissue heterogeneity mapping
• Longitudinal single-cell profiling in developmental and stem cell biology
• High-content drug screening and biomarker discovery

Conclusion

The combination of nanoPOTS nanodroplet processing, TMT10plex multiplexing and real-time search enabled SPS MS3 on an Orbitrap Eclipse Tribrid platform provides a robust, high-throughput workflow for single-cell proteomics. This approach achieves deep protein coverage, accurate quantification and reliable cell classification, establishing a foundation for broad adoption in research and clinical applications.

References

1. Zhu Y et al Angewandte Chemie International Edition 57 12370 2018
2. Budnik B et al Genome Biology 19 161 2018
3. Erickson BK et al Journal of Proteome Research 18 1299 2019