Enabling scalable single-cell proteomics by utilizing the unique analytical properties of the Evotip Pure with new Whisper Zoom methods

Significance of the Topic

Single-cell proteomics is emerging as a critical tool for understanding cellular heterogeneity. Achieving scalable workflows with high sensitivity and robustness is essential to analyze minute protein quantities and expand throughput in research and clinical applications.

Objectives and Study Overview

This study introduces Whisper Zoom methods coupled with the Evotip Pure device to enable high-throughput, reproducible single-cell proteomics. The goals include evaluating performance gains over legacy workflows across different sample loads and assessing robustness in multi-lab settings.

Methodology and Instrumentation

• Sample Preparation and Workflow: Single-cell equivalents (250 pg HeLa digest) and higher loads (5–200 ng) were captured on disposable Evotip Pure trap columns for retention and minimal loss.
• Chromatography: Four Whisper Zoom methods (20, 40, 80, 120 samples per day) operated at 200 nl/min using an Aurora Rapid 75 column.
• Mass Spectrometry: Analyses performed on a timsTOF Pro 2 with dia-PASEF acquisition and library-free DIA-NN data processing against the human Swiss-Prot proteome.
• Robustness Assessment: Inter- and intra-instrument reproducibility tested across five Evosep One systems internally and three external labs.

Key Results and Discussion

• Performance Improvement: Whisper Zoom 80 SPD doubled signal intensity over legacy methods, while 120 SPD achieved sharper peaks (average 1.5 s FWHM) and high precursor identification rates.
• Reproducibility: Retention time variability remained below 2.6 s standard deviation across 340 runs and multiple instruments.
• Carryover: Single-cell-level carryover was minimal (0.6% following plasma, 0.02% following HeLa), supporting scalable sample sequences with low contamination.
• Sensitivity and Saturation: Saturation of identifications observed at 10 ng for 120 SPD, 50 ng for 40/80 SPD, and 200 ng for 20 SPD; dynamic range preserved down to sub-nanogram loads.
• Sample Storage: Evotip storage maintained over 90% identifications after 48 h and 75% after 72 h; addition of DDM surfactant enhanced low-load identifications without affecting storage integrity.

Benefits and Practical Applications

• High Throughput: Up to 120 samples per day with consistent sensitivity and precision.
• Scalability: Suitable for low-volume single-cell analyses and larger sample loads without method adjustments.
• Robustness and Reproducibility: Low inter-instrument variability and carryover support large-scale studies and multi-site deployments.
• Sample Integrity: The disposable trap column design reduces losses and simplifies workflow handling.

Future Trends and Opportunities

• Integration with Emerging Cell Sorting: Combining high-sensitivity proteomics with automated cell dispensers for streamlined single-cell studies.
• Expansion to Clinical Biomarkers: Scalable workflows could enable routine single-cell proteome profiling in diagnostics and personalized medicine.
• Method Optimization: Further refinement of surfactants and trap chemistries to enhance low-abundance protein coverage.
• Automation and Data Analysis: Coupling automated sample handling with advanced AI-driven data processing to accelerate discovery.

Conclusion

The novel Whisper Zoom methods with Evotip Pure significantly advance single-cell proteomics by delivering high sensitivity, robustness, and throughput. Their proven reproducibility across instruments and labs positions them as a versatile platform for research and clinical applications.