Label-free DIA-based workflow for single-cell proteomic analysis on an Orbitrap Ascend Tribrid mass spectrometer

Significance of the Topic

Single-cell proteomics represents a frontier in analytical chemistry, enabling detailed characterization of cellular heterogeneity, signaling pathways and rare cell populations. Overcoming challenges of sensitivity, dynamic range and throughput is critical for applications across biomedical research, drug discovery and quality control.

Objectives and Study Overview

This study evaluates a label-free data-independent acquisition (DIA) workflow combining the Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer with the Vanquish Neo UHPLC system. The main goals are to demonstrate high sensitivity for low-input samples, achieve 50 samples per day (SPD) throughput and assess performance on HeLa peptide dilutions and true single-cell preparations.

Methodology

A DIA-based method was developed for 25-minute LC–MS runs, yielding 50 SPD. Key steps include:

• Sample preparation: Thermo Pierce HeLa protein digest diluted to inject loads from 0.05 to 10 ng; single HeLa cells sorted via CellenONE onto proteoChip LF 48 and processed in 384-well format.
• Chromatography: Vanquish Neo UHPLC with Aurora UltiMate 25 cm × 75 µm column at 300 nL/min.
• Mass spectrometry: Orbitrap Ascend Tribrid with FAIMS Pro Duo interface, full MS at 120 000 resolution (m/z 400–800), DIA windows sized 40 m/z (>1 ng) or 50 m/z (<1 ng), HCD collision energy 28%, cycle time ~1 s.
• Data processing: Library-free searches on Spectronaut 18 and Thermo Proteome Discoverer 3.1 with CHIMERYS algorithm; 1% FDR and match-between-runs enabled.

Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system with EASY-Spray ion source
• Orbitrap Ascend Tribrid mass spectrometer
• FAIMS Pro Duo interface
• CellenONE single-cell dispenser and proteoChip LF 48 plates

Main Results and Discussion

The workflow demonstrated robust identification depth and reproducibility across low input and single-cell samples:

• HeLa peptide dilution series: Protein group IDs rose from ~1 300 at 0.05 ng to over 5 000 at 10 ng; peptide IDs increased accordingly, illustrating linear sensitivity.
• Single-cell analysis: Spectronaut searches identified ~865 protein groups from one cell, ~3 100 from five cells and ~4 300 from ten cells. CHIMERYS-powered searches further increased single-cell IDs to ~1 250.
• DIA library size impact: Larger libraries enhanced IDs for low-input runs but introduced slight variability in quantitation CVs.
• Throughput: The 50 SPD method maintained identification depth and quantitative precision (CV <20%) across all sample loads.

The combination of low-nanoflow chromatography, FAIMS interface and rapid Orbitrap scanning provides marked gains in sensitivity and throughput, addressing key limitations of conventional single-cell proteomics.

Practical Benefits and Applications

Implementing this workflow in research and industrial settings can:

• Enable high-throughput profiling of cellular subpopulations in disease and developmental studies.
• Support quality control in biomanufacturing by monitoring single-cell productivity and stress responses.
• Facilitate drug screening campaigns with fast sample turnover and deep coverage.

Future Trends and Applications

Emerging directions include:

• Integration of microfluidic sample preparation for full automation and reduced losses.
• Advanced ion mobility and AI-based search algorithms to push coverage below current sensitivity limits.
• Multiplexed DIA strategies for simultaneous analysis of cell populations with minimal instrument downtime.

Conclusion

The described label-free DIA workflow on Orbitrap Ascend Tribrid coupled with Vanquish Neo UHPLC achieves high sensitivity for single-cell and low-input proteomics at 50 samples/day. It offers a balanced solution of depth, throughput and quantitative precision suitable for diverse applications in research and industry.

References

1. Runseng Z, Matzinger M, Mayer RL, Valenta A, Sun X, Mechtler K. A High-Sensitivity Low-Nanoflow LC-MS Configuration for High-Throughput Sample-Limited Proteomics. Anal. Chem. 2023;95(51):18673–18678.