High-resolution DIA proteomics workflow for single-cell samples on the Orbitrap Astral mass spectrometer

Importance of the topic

Single-cell proteomics enables detailed exploration of biological heterogeneity by profiling the proteome at the individual cell level. High-sensitivity analysis with preserved throughput and chromatographic performance is essential to generate high-quality data and uncover subtle molecular differences across cells.

Objectives and study overview

This study aimed to assess proteome coverage and sample throughput for single-cell samples using both library-free and library-based data-independent acquisition (DIA) workflows on the Thermo Scientific Orbitrap Astral mass spectrometer. A dilution series of HeLa protein digest ranging from 5 ng to 50 pg was used to evaluate sensitivity, precision, accuracy and linearity before analyzing individual cells.

Methodology and instrumentation

The single-cell proteomics workflow combined high-performance chromatography and advanced mass spectrometry:

• UHPLC system: Thermo Scientific Vanquish Neo with reduced flow rates and precise gradient formation
• Chromatography column: Thermo Scientific μPAC Neo 50 cm micro-pillar array C18 column at 50 °C
• Ion mobility interface: Thermo Scientific FAIMS Pro Duo to reduce background and enhance signal‐to‐noise
• Mass spectrometer: Thermo Scientific Orbitrap Astral combining high‐resolution Orbitrap full scans (240 k) with high‐speed Astral MS/MS
• Data acquisition: DIA windows tailored by sample load (5–20 m/z windows; 10–60 ms injection times)
• Data analysis: Biognosys Spectronaut 18 (library-free directDIA and library-based) and Thermo Scientific Proteome Discoverer 3.1 with CHIMERYS

Key results and discussion

In the dilution series, library-free processing of triplicates quantified over 2,700 protein groups at 50 pg and over 6,650 at 5 ng. Processing all files together increased identifications by up to 70% at low loads while maintaining precision (median CV <10% for loads ≥250 pg) and linearity across a 1:100 ratio range.

For single‐cell HeLa samples using an 18 min cycle (72 samples/day), individual analyses yielded on average >3,400 protein groups and 15,300 peptides per cell. Joint processing of 12 cells increased coverage to >4,250 protein groups and >23,550 peptides. Incorporating a library from 20 cells further raised quantified protein groups to 5,405.

Benefits and practical applications

The Orbitrap Astral workflow delivers robust, reproducible and rapid deep proteome coverage from limited sample amounts. The FAIMS Pro interface significantly improves spectral quality by suppressing background ions. High‐precision and accurate quantitative performance enables reliable biomarker discovery and pathway analysis in single‐cell studies.

Future trends and potential applications

Advances in single-cell proteomics will focus on even higher throughput, refined library‐free algorithms, integration with spatial imaging and application to diverse cell types and complex tissues. Continued improvements in chromatography, ion mobility and data processing will expand the scope of biological questions addressable at single‐cell resolution.

Conclusion

The combination of the Vanquish Neo UHPLC system, μPAC Neo column, FAIMS Pro interface and Orbitrap Astral mass analyzer establishes a new standard for sensitive, high‐throughput single‐cell proteomics. The workflow achieves deep proteome coverage, excellent quantitative precision and accuracy, and versatile library‐free and library‐based DIA strategies.