B-146 Unlocking the potential of large-cohort proteomics studies with the Orbitrap Astral mass platform
Authors: Xuan Y , Pashkova A , Zeller M , Wirth C T , Grote J , Arrey T N , Damoc E N , Hart B J 
Published: October 2024

Abstract

Background
Large-cohort proteomics analysis using mass spectrometry is a powerful approach to discover and validate new biomarkers. In combination with clinical data and computational analysis, large-cohort proteomics study brings opportunities in improving early diagnosis, refining patient stratification, and predicting/monitoring treatment response. Yet, to achieve meaningful biological insights in large-cohort studies, robust, reproducible, and comprehensive proteome profiling in a high-throughput manner still remains challenging. Here, we use a high-resolution accurate mass (HRAM) Oribtrap Astral platform to enable high-quality and robust protein quantification across thousands of LC-MS/MS analyses. With a throughput of 100 samples/day, we can reproducibly profile ∼9000 proteins from human cell line and ∼800 proteins from undepleted plasma across multiple instruments and more than 10 consecutive days in a 24/7 operation mode.

Methods
Experiments were performed on multiple Orbitrap Astral mass spectrometers with and without FAIMS Pro interface. Chromatographic separations were performed using a trap/elute method on Vanquish Neo system at a 11-minute gradient and a flow rate of 1ul/min, resulting in a throughput of 100 samples/day. Undepleted plasma digest was analyzed in a 24/7 mode for &gt; 1000 injections on each LC-MS/MS setup. In addition, HeLa digest as quality control (QC) was analyzed every 12hours. The Oribtrap Astral platform was operated in data-independent acquisition (DIA) mode using a full scan with m/z 380-980, and DIA MS/MS scans with an isolation window of 2Th. Resulting DIA raw files were automatically transferred and processed using Chimerys in a beta version of Proteome Discoverer software.

Results
Multiple LC-MS/MS systems were operated in DIA mode either with or without FAIMS Pro device in a 24/7 operation mode at a throughput of 100SPD. Undepleted plasma digest was analyzed with &gt;1000 injections on each LC-MS/MS setup. To monitor the baseline performance, HeLa digest served as QC were analyzed periodically every 12 hours with 3 technical replicates each time. To effectively manage and analyze these thousands of data files generated, we developed an automated data transfer and analysis pipeline. Automatically, the resulting DIA raw data files were immediately transferred to a server, then processed by state-of-the-art intelligent search algorithm Chimerys. Benefiting from the ultra-high scan speed of up to 200Hz on the Orbitrap Astral mass spectrometer, a much narrower isolation window width of 2Th was applied in the DIA method, comparing to 10-20Th on the classic DIA method. As a result, ∼9000 proteins from HeLa digest and ∼800 proteins from undepleted plasma digest were identified within only a 11-minutes gradient, respectively. More than 80% of the plasma proteins were reproducibly identified and quantified from all the runs on each LC-MS/MS setup, indicating a great reproducibility from run-to-run longitudinally. Stable and robust peptide quantitation was observed by extracting peptides with high, medium, and low abundant across the runs. Importantly, QC showed no performance degradation throughout the entire study, indicating high robustness of the entire LC-MS/MS setup.

Conclusions
Orbitrap Astral mass platform can comprehensively analyze the proteome of &gt;1000s of sample robustly and reproducibly in a high-throughput manner, addressing the needs in large-cohort studies.