Optimized one-pot single-cell proteomics workflow

Significance of the Topic

Single-cell proteomics overcomes the averaging effects of bulk-cell analysis and reveals the diversity of protein expression and post-translational modifications at the individual cell level. This level of detail is crucial for understanding heterogeneous cell populations in health and disease, identifying rare cell subsets, and gaining insights into cellular processes that cannot be inferred from nucleic acid measurements alone.

Objectives and Overview of the Study

This application note describes the development of an optimized one-pot workflow for label-free single-cell proteomics that maximizes both proteome coverage and sample throughput. The study integrates advanced cell isolation, sample preparation, chromatography, gas-phase fractionation, mass spectrometry, and AI-driven data analysis into a unified platform. A novel wide window acquisition strategy is introduced to further enhance peptide identification.

• Develop an automated one-pot sample preparation protocol in submicroliter volumes
• Implement wide window acquisition WWA on Orbitrap Exploris 480 with FAIMS Pro for sensitive data acquisition
• Apply CHIMERYS AI-based search for deep proteome coverage from chimeric spectra

Methodology and Instrumentation

Sample preparation and cell isolation were carried out using the following key components:

• Cell Dispensing and Isolation: cellenONE instrument with proteoCHIP plates for image-based single-cell isolation at rates up to 40 cells per minute, including automated hydration and 5% DMSO supplementation to prevent evaporation and improve peptide recovery
• Sample Lysis and Digestion: One-pot digestion in 384-well plates using trypsin, DDM surfactant, TEAB buffer, and ProteaseMAX enhancer performed at 50 °C with sequential enzyme additions and periodic hydration
• Chromatography: Thermo Scientific Vanquish Neo UHPLC system coupled to µPAC Gen2 HPLC columns for ultralow flow separations (100–1,000 nL/min) with high resolution and retention-time precision
• Gas-Phase Fractionation and MS: FAIMS Pro interface for removal of singly charged background ions combined with an Orbitrap Exploris 480 mass spectrometer for sensitive MS and MS2 acquisition
• Data Analysis: Thermo Scientific Proteome Discoverer software with the CHIMERYS intelligent search node and Percolator FDR control for AI-driven identification of up to eleven coisolated peptides per spectrum

Main Results and Discussion

The optimized workflow achieved robust identification of over 1,300 proteins and 4,500 peptides from a single-cell equivalent digest. Key findings include:

• Sequential trypsin additions, periodic hydration, and DMSO supplementation increased protein IDs by 527 compared to single-addition protocols while retaining over 90% overlap with the original dataset
• µPAC Neo columns doubled chromatographic resolution and sensitivity relative to earlier versions, enabling sharper peaks and greater depth in low-flow methods
• Wide window acquisition with an 8 to 12 m/z isolation window optimized PSM and protein yield for 250 pg HeLa digest, reaching a plateau of 600 proteins and continued gains in PSMs as window width increased
• CHIMERYS AI-based spectral prediction doubled peptide and protein identifications versus conventional search engines (MS Amanda and SEQUEST) under equivalent gradient conditions
• Dynamic range spanned four to five orders of magnitude, demonstrating sensitive detection of both high-abundance contaminants and low-abundance histone peptides without the need for carrier channels

Benefits and Practical Applications of the Method

This integrated workflow offers:

• High sensitivity and reproducibility in single-cell proteomics without reliance on carrier proteome boosting
• Automated, low-volume sample preparation compatible with rare cell types and low cell concentrations
• Enhanced throughput via shorter chromatographic gradients and streamlined data acquisition modes
• Deep proteome coverage with minimal missing values, enabling robust comparative studies and biomarker discovery

Future Trends and Applications

Emerging applications include profiling individual cells in tumor microenvironments to study cancer initiation and metastasis, mapping proteomic heterogeneity in brain tissue and organoids to investigate neural development, and identifying early disease biomarkers for precision medicine. Integration with multiplexed quantitation and PTM-focused workflows promises further insights into cell-to-cell variability in signaling and regulatory networks.

Conclusion

The optimized one-pot single-cell proteomics platform combining cellenONE isolation, µPAC Neo chromatography, FAIMS-enhanced Orbitrap Exploris 480 acquisition, and CHIMERYS AI-driven analysis delivers unprecedented sensitivity, depth, and throughput in label-free single-cell studies. This workflow paves the way for routine high-impact biological and biomedical investigations at the single-cell level.

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