Orbitrap Astral mass spectrometer allows comprehensive proteome coverage at the single-cell level

Importance of the topic

Single-cell proteomics unravels heterogeneity at the level of individual cells, providing insights into cell development, tumor diversity, disease progression and drug response. By moving beyond bulk analyses, this field enables discovery of rare cell states and potential therapeutic targets.

Objectives and overview

This work demonstrates a streamlined workflow for high-throughput, sensitive single-cell proteomics using the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer. It evaluates proteome coverage in a HeLa digest dilution series (50–10 000 pg) and real HeLa single cells isolated with the CellenONE™ system on proteoCHIP EVO 96 and standard 96/384-well plates. Data-independent acquisition (DIA) methods are compared in library-free and library-based modes under a rapid 14 min LC gradient, enabling 80 samples per day.

Methodology

A one-pot sample preparation integrates automated single-cell sorting, lysis and digestion in nanoliter volumes using a DDM/TEAB/trypsin master mix within proteoCHIP EVO 96 or 96/384-well plates. Controlled incubation at 50 °C and 85 % humidity ensures efficient digestion, followed by quenching with 0.1 % TFA/1 % DMSO and direct injection.

Instrumentation used

• Sample handling: CellenONE™ system with proteoCHIP EVO 96 or standard plates
• Chromatography: Vanquish Neo UHPLC, IonOpticks Aurora Elite 15 cm×75 µm C18 column, 14 min gradient at 0.2 µL/min (80 SPD)
• Mass spectrometry: Orbitrap Astral MS with FAIMS Pro Duo, 240 k MS1 resolution, >200 Hz MS/MS, operated in DIA mode
• Data analysis: Spectronaut 18 (library-free and library-based DIA) and Proteome Discoverer with CHIMERYS™

Main findings and discussion

In the HeLa digest dilution series, library-free DIA identified >4 200 proteins from 50 pg and >5 500 from 250 pg; library-based DIA increased identifications to >6 100 and >7 200, respectively, with median CV < 10 %. Analysis of 222 single HeLa cells yielded on average 5 977 proteins (34 894 peptides, R = 0.93–0.98, dynamic range > 5 orders) in 384-well plates; proteoCHIP EVO 96 delivered 4 589 proteins per cell; 96-well plates yielded 6 556 proteins. Blank controls showed minimal carryover (< 180 proteins). Membrane proteins comprised ~22 % of identifications.

Benefits and practical applications

• Automated, low-volume, lossless preparation reduces contamination and handling steps
• High throughput (80 samples/day) with a rapid 14 min LC gradient
• Deep proteome coverage at single-cell and sub-nanogram levels
• Robust reproducibility (CV < 10 %) and wide dynamic range (> 5 orders)

Future trends and applications

Emerging directions include integration of carrier or booster channels, further FAIMS CV optimization and AI-driven data analysis. Applications will expand to primary clinical specimens, spatial proteomics, multi-omics integration and profiling of rare cell populations in complex tissues.

Conclusion

The described workflow combines microfluidic sorting, ultrafast LC, Orbitrap Astral MS and DIA strategies to achieve reproducible, comprehensive proteome profiling at the single-cell level with high sensitivity, throughput and quantitative accuracy.

Reference

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