Pushing the boundaries for robust and high-throughput single cell analysis with Whisper Flow Technology powered by dia-PASEF

Significance of the topic

Single-cell proteomics unlocks insights into cellular heterogeneity that bulk analyses cannot provide.
The challenge lies in achieving sufficient sensitivity, reproducibility and throughput to analyze hundreds of individual cells robustly.
The Whisper Flow Technology integrated with dia-PASEF on the timsTOF SCP platform addresses these challenges by combining optimized sample handling, chromatography and mass spectrometry.

Objectives and Study Overview

This work aims to develop a streamlined, high-throughput workflow for single-cell proteomics that delivers deep proteome coverage and high reproducibility.
Key goals include mapping proteomes of individual HeLa and A549 cells, scaling throughput from 40 to 80 single cells per day, and minimizing sample loss during transfer.
The study integrates automated cell sorting, prototype proteoCHIP digestion, direct peptide transfer via centrifugation into Evotips, and robust chromatographic gradients with dia-PASEF acquisition.

Methodology and Instrumentation

Sample preparation and handling steps:

• Cell sorting by size using the cellenONE platform into wells of a proteoCHIP prototype.
• On-chip lysis, digestion and peptide generation without manual pipetting.
• Centrifugation-driven transfer of peptides from the proteoCHIP into Evotips to enhance reproducibility.
• Storage of loaded Evotips at room temperature on the instrument for up to 2.5 days without detectable loss in proteome depth.

Chromatography and acquisition strategies:

• Use of Whisper 40 samples per day (SPD) and Whisper 80 SPD methods on an Evosep One system.
• Aurora Elite column (100 nl/min flow) for 40 SPD; Aurora Rapid 75 column for 80 SPD to increase throughput.
• Data-independent acquisition with parallel accumulation–serial fragmentation (dia-PASEF) on a timsTOF SCP mass spectrometer.
• Data processing with DIA-NN 1.8.1 using a comprehensive spectral library generated from deeply fractionated human cell lines.

Instrumentation

• cellenONE single-cell dispenser (Cellenion)
• Prototype proteoCHIP for on-chip digestion
• Evotips sample cartridges (Evosep Biosystems)
• Evosep One liquid chromatography system
• Aurora Elite and Aurora Rapid 75 nano-flow columns (IonOpticks)
• timsTOF SCP mass spectrometer (Bruker)
• DIA-NN software for data analysis

Main Results and Discussion

Proteome depth and reproducibility:

• Average identification of ~3,500 protein groups per single HeLa cell using Whisper 40 SPD, with almost 5,500 proteins across all replicates.
• High retention-time stability (standard deviation 11–13 s) and sharp chromatographic peaks (FWHM 2.5–4.1 s) at 100 nl/min.
• Centrifugation-based transfer yielded tighter distributions of identified precursors compared to manual pipetting.

Sensitivity and scalability:

• Dilution series of HeLa tryptic peptides (62.5 pg to 32 ng) demonstrated robust protein identification at sub-nanogram loads.
• Whisper 80 SPD method on Aurora Rapid 75 column enabled analysis of 80 A549 single cells per day, averaging ~1,400 proteins per cell.

Biological insights:

• Principal component analysis separated HeLa cells sorted by diameter (18–20, 21–23, 24–26, 27–30 µm).
• ANOVA identified ~2,648 proteins significantly regulated across size groups, including metabolic enzymes, stress response proteins and nuclear factors.

Benefits and Practical Applications

• High-content proteome maps from individual cells without loss of depth during storage or transfer.
• Increased throughput up to 80 cells per day to support large-scale studies of cellular heterogeneity.
• Reproducible and automation-friendly workflow reduces manual steps and variability.
• Applicable to basic research, clinical proteomics, drug discovery and quality control in biomanufacturing.

Future Trends and Potential Uses

• Further integration with robotics and microfluidics to push throughput beyond 100 cells/day.
• Extension to tissues, rare cell types and spatially resolved single-cell proteomics.
• Combining single-cell proteomics with transcriptomics and metabolomics for multi-omics profiling.
• Enhanced data analysis pipelines leveraging machine learning for deeper insights into cell states and signaling networks.

Conclusion

The Whisper Flow Technology powered by dia-PASEF on the timsTOF SCP establishes a robust, high-throughput single-cell proteomics workflow.
Key innovations in sample transfer, chromatography and acquisition deliver deep proteome coverage, high reproducibility and scalable throughput.
This platform opens new opportunities for comprehensive studies of cellular heterogeneity in research and applied settings.