Single-Shot LC-MS Workflow for Comprehensive Proteome Identification on an Orbitrap Astral Mass Spectrometer

Importance of the Topic

Modern proteomics demands comprehensive coverage of complex biological samples with high throughput and reproducibility. Single-shot LC-MS workflows eliminate time-consuming prefractionation, reduce sample loss, and streamline analysis for large-scale studies. The Orbitrap Astral mass analyzer enhances scan speed and sensitivity, enabling deep proteome coverage in one-hour gradients.

Study Goals and Overview

This study presents an end-to-end single-shot LC-MS workflow integrating the Thermo Scientific Orbitrap Astral mass spectrometer and Vanquish Neo UHPLC system with label-free data-independent acquisition. Eleven commonly used human cell lines were analyzed in biological triplicates to evaluate proteome depth, quantitation precision, and sample throughput.

Methodology and Instrumentation

• Sample preparation: Eleven cell lines were cultured, lysed, and quantified by BCA assay. Fifty micrograms of protein were processed on the AccelerOme platform for reduction, alkylation, in-solution trypsin digestion, and peptide cleanup.
• Chromatography: Cell line peptides (500 ng) were separated on a μPAC Neo 50 cm C18 column using a 60-minute gradient at 60 samples per day (SPD). A PepMap trap-elute setup with a 15 cm analytical column and 5 mm trap column supported throughput tests at 30–300 SPD.
• Mass spectrometry: The Orbitrap Astral MS acquired full scans at 240 000 resolution (FWHM) with normalized AGC target 500% and 3.5 ms injection time. DIA used 2 Th isolation windows over 380–980 m/z and HCD MS/MS at 150–2000 m/z with 40% RF lens.
• Data processing: Proteome Discoverer 3.1 with CHIMERYS 2.5.15 and directDIA searched against the human UniProt database (including isoforms), applying a 1% false discovery rate filter for peptides and proteins.

Key Results and Discussion

• Proteome coverage: Up to 12 000 protein groups and 160 000 peptides were identified for the PEO1 ovarian cell line using the one-hour DIA method.
• Reproducibility: Biological triplicates across eleven cell lines achieved a median coefficient of variation of approximately 10% at the protein group level.
• Throughput vs precision: Standard HeLa digest analyses at 60, 100, and 180 SPD yielded median protein CVs of 4.0%, 4.3%, and 5.4%, and peptide CVs of 11.0%, 11.3%, and 13.2% respectively, demonstrating high quantitation accuracy at elevated throughput.
• Efficiency: The single-shot approach replaced traditional 24–48 fraction workflows, enabling processing of over 5000 samples per month per instrument without sacrificing proteome depth.

Benefits and Practical Applications

The described workflow delivers robust, reproducible, and high-depth proteome profiling suitable for large-scale biological experiments, clinical biomarker discovery, quality control in biotechnology and pharmaceutical research. Label-free DIA simplifies sample preparation and minimizes inter-run variability.

Future Trends and Potential Applications

• Integration of automation and robotics to scale sample preparation and instrument handling.
• Application of artificial intelligence and machine learning to enhance peptide identification and quantitative accuracy.
• Extension to targeted DIA, post-translational modification mapping, and single-cell proteomics.
• Multi-omics integration for comprehensive systems biology studies and precision medicine.

Conclusion

This single-shot LC-MS workflow, combining the Orbitrap Astral analyzer with Vanquish Neo UHPLC and AccelerOme sample preparation, achieves deep, reproducible proteome coverage at high throughput. It provides a versatile platform for comprehensive proteomic analysis in research and industrial environments.

References

• 1. Wang H, Yang Y, Li Y, Bai B, Wang X, Tan H, Liu T, Beach TG, Peng J, Wu Z. Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome. J Proteome Res. 2015;14:829-38.
• 2. Meier F, Geyer PE, Virreira Winter S, Cox J, Mann M. BoxCar acquisition method enables single-shot proteomics at a depth of 10 000 proteins in 100 minutes. Nat Methods. 2018;15:440-448.
• 3. Stejskal K, Op De Beeck J, Matzinger M, Dürnberger G, Boychenko A, Jacobs P, Mechtler K. Deep proteome profiling with reduced carryover using superficially porous microfabricated nanoLC columns. Anal Chem. 2022;94:15930-15938.