Deeper proteome coverage and faster throughput for low input samples on the Thermo Scientific Orbitrap Astral mass spectrometer

Significance of the Topic

Advances in mass spectrometry for proteomics have driven interest in analyzing ever smaller sample quantities, including individual cells. High sensitivity, deep proteome coverage, and rapid throughput are essential to capture biological heterogeneity and achieve statistically robust data from large cohorts of low-input samples.

Objectives and Study Overview

This study evaluates the performance of the Thermo Scientific Orbitrap Astral mass spectrometer for low-input proteomics. The goal is to compare library-free (directDIA) and library-based data-independent acquisition (DIA) methods across sample loads from 50 pg to 10 ng of HeLa protein digest. Key performance metrics include depth of proteome coverage, quantitative precision, and sample throughput at 80 samples per day.

Methodology and Workflow

The workflow uses a Thermo Scientific Vanquish Neo UHPLC system coupled to a 50 cm µPAC Neo Low Load column and a FAIMS Pro interface for enhanced signal-to-noise. Samples of Pierce HeLa protein digest standard were prepared at defined dilutions and analyzed using an 18-min injection-to-injection cycle (8-min gradient plus wash/equilibration). DIA parameters were optimized: a 20 m/z isolation window and extended injection times (up to 50 ms) on the Astral MS2 analyzer for sub-nanogram loads. Data processing employed Spectronaut 17 directDIA and library-based workflows against the human UniProt database with 1% FDR and match-between-runs enabled.

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system (Pump/Autosampler, Column Compartment)
• µPAC Neo Low Load HPLC column, 50 cm C18 micropillar array
• Thermo Scientific FAIMS Pro interface
• Thermo Scientific Orbitrap Astral mass spectrometer with Orbitrap MS1 and Astral MS2 analyzers
• Spectronaut 17 software (Biognosys AG)

Main Results and Discussion

• Optimal DIA window of 20 m/z yielded highest IDs for 250 pg digest (~4,000 proteins, ~15,900 peptides).
• Extended Astral MS2 injection times (40–50 ms) improved identifications at low loads—up to ~2,600 proteins from 50 pg.
• Library-free directDIA achieved ~3,300 proteins at 250 pg; library-based DIA using a 250 pg–10 ng spectral library reached ~5,744 proteins at 250 pg.
• At 10 ng load, ~6,594 protein groups and ~46,900 peptides were identified in directDIA; library-based results plateaued above 5 ng.
• Quantitative precision was high: median CV of 11.9% at 250 pg and 8.9% at 10 ng, with over 60% of proteins below 20% CV at single-cell equivalent loads.

Practical Benefits and Applications

The Orbitrap Astral platform delivers unmatched sensitivity and throughput for low-input proteomics, enabling near single-cell depth of coverage in an 80 SPD workflow. Its combination of high-resolution MS1, high-speed MS2, FAIMS background reduction, and micropillar chromatography provides robust, high-precision quantitation suitable for biomarker discovery, cell-type profiling, and QA/QC in industrial analytics.

Future Trends and Potential Applications

Emerging directions include integration of in-silico spectral libraries powered by machine learning, further acceleration of chromatographic and MS acquisition speeds, and expansion into multi-omic single-cell platforms. Clinical and translational research will benefit from deeper, high-throughput profiling of minimal sample inputs.

Conclusion

The Thermo Scientific Orbitrap Astral mass spectrometer, combined with Vanquish Neo UHPLC, µPAC Neo columns, and FAIMS Pro, establishes a new benchmark for deep, rapid, and precise proteome analysis of low-input samples. Both library-free and library-based DIA approaches demonstrate unprecedented performance down to 50 pg, paving the way for routine single-cell proteomics.