A Benchmarking Workflow for High-Throughput DIA Label-Free Quantification using a Novel High-Resolution Accurate Mass Platform

Importance of the topic

Label free quantitation using data independent acquisition on a high resolution accurate mass platform enables rapid and deep proteome profiling. The ability to quantify thousands of proteins across a wide dynamic range is essential for applications in biomarker discovery quality control and systems biology.

Goals and study overview

The study aims to benchmark a novel Orbitrap Astral mass spectrometer for label free quantitation under diverse conditions. Three proteome digests mixing human yeast and E coli at defined ratios were analyzed across varying sample loads gradient lengths and acquisition parameters to assess quantitation depth accuracy and precision.

Methodology and instrumentation

Sample preparation followed bottom up proteomics standards with a micropillar array column for reproducible separation. Ultrahigh performance liquid chromatography was performed on a Vanquish Neo UHPLC in direct injection or trap and elute modes. The Orbitrap Astral instrument combined a quadrupole for precursor selection an Orbitrap HRAM analyzer and a novel Astral mass analyzer to deliver up to 200 hertz data acquisition in DIA mode with 2Th isolation windows. Data processing employed Spectronaut 17 directDIA and Proteome Discoverer 3.1 with CHIMERYS search algorithm at 1 percent false discovery rate.

Main results and discussion

Over thirteen thousand proteins were identified and quantified across triplicate runs of mixed proteomes from 500 nanogram loads. Quantitative precision was high with a median coefficient of variation of 4.7 percent and 87 percent of proteins showing CV below 20 percent. Dynamic range extended over five orders of magnitude enabling reliable quantitation from low abundance hundred copies per cell to high abundance hundred thousand copies per cell. Throughput studies with 180 100 and 60 samples per day modes demonstrated consistent accuracy independent of gradient length or sample load. Single peptide quantitation showed performance comparable to multi peptide based measurements.

Benefits and practical applications

• High sensitivity and dynamic range for comprehensive proteome coverage
• Rapid acquisition supports high sample throughput
• Robust quantitation at low sample loads ideal for limited material
• Reliable single peptide quantitation enhances detection of low abundance proteins

Future trends and possible applications

Further integration of high rate acquisition with advanced machine learning based data analysis may advance real time quality control and enable single cell proteomics. Shorter gradients and automated sample handling will enhance throughput for clinical and industrial workflows. Development of more flexible DIA schemes may optimize performance for targeted applications.

Conclusion

The Orbitrap Astral mass spectrometer delivers ultrafast data acquisition high sensitivity and wide dynamic range supporting accurate label free quantitation across diverse conditions. Its performance opens new opportunities in high throughput proteomics systems biology and biomarker research.

Reference

Ghaemmaghami S Huh WK Bower K et al Global analysis of protein expression in yeast Nature 425 737–741 2003