Ultra-high sensitive Single Cell Proteomics on the timsTOF Ultra

Significance of the Topic

Single-cell proteomics addresses the challenge of cellular heterogeneity by profiling protein expression on an individual cell basis. Achieving comprehensive proteome coverage at this scale is crucial for understanding biological processes in health and disease, enabling precise characterization of rare cell populations and improving biomarker discovery.

Study Objectives and Overview

This work demonstrates an ultra-high-sensitivity workflow for single cell proteomics using the timsTOF Ultra mass spectrometer. The study compares direct injection from vials versus an automated “dissolve” function for lyophilized samples, evaluates short (10 min) versus longer (22 min) gradients, and applies the method to K562 peptide dilutions, HeLa cell pools (1–20 cells), and primary human immune cells for robust performance assessment.

Methodology and Instrumentation

The protocol integrates automated single cell isolation with the cellenONE® platform and sample preparation in the proteoCHIP LF 48 format to minimize losses. Lyophilized peptide or cell digest spots are resuspended immediately before injection using a novel autosampler “dissolve” function on the nanoElute 2 system. Analysis is performed in dia-PASEF acquisition mode at flow rates of 250 nL/min, using either a 10 min (80 SPD) or 22 min (32 SPD) active gradient on a 5 cm Aurora Rapid column.

Instrumentation Used

• Cell sorting and dispensing: cellenONE® (Cellenion)
• Sample handling: proteoCHIP LF 48 (Bruker)
• Autosampler: nanoElute 2 CTC with dissolve function (Bruker)
• Liquid chromatography: nanoElute 2 system, 250 nL/min flow
• Mass spectrometry: timsTOF Ultra operated in dia-PASEF mode (Bruker)
• Data analysis: Spectronaut 18 directDIA+ (Biognosys)

Main Results and Discussion

K562 peptide dilution series achieved identification of thousands of protein groups down to 15.6 pg loads, both with and without reference-run evaluation. HeLa single cell analyses yielded on average 1,700 protein groups per cell, with coefficient of variation (CV) below 20% across replicates. Shorter 10 min gradients delivered comparable depth to 22 min runs, supporting high throughput without major sensitivity loss. Application to FACS-sorted human PBMC subtypes (CD4+, CD8+, CD14+, CD19+) demonstrated clear separation in PCA and distinct clustering in abundance heatmaps, confirming the method’s capacity to resolve cell-type-specific proteomes.

Practical Benefits and Applications

• Hands-free, pipetting-free workflow reduces sample loss and contamination risk.
• High sensitivity and reproducibility support routine analysis of rare or limited cell populations.
• Flexible gradient options enable balancing throughput and depth for different study designs.
• DirectDIA+ analysis simplifies data processing and boosts identification rates without extensive method optimization.

Future Trends and Potential Applications

Ongoing developments may include integration with multi-omics platforms (e.g., transcriptomics, metabolomics), further optimization of ion transfer and acquisition schemes, and AI-driven data interpretation. Scaling to higher-density sample formats and automated multiplexing will expand application in clinical research, drug discovery, and personalized medicine.

Conclusion

This workflow combining cellenONE isolation, proteoCHIP preparation, automated sample dissolution, and timsTOF Ultra dia-PASEF analysis achieves unprecedented sensitivity and reproducibility in single cell proteomics. It provides a robust, high-throughput solution for in-depth proteome profiling of individual cells, opening new avenues in biological research and precision diagnostics.

Reference

No formal references were provided in the source material.