High throughput proteome and phosphorpoteome sample processing coupled to fast gradient DIA

• Published in: Journal of biomolecular techniques Vol. 31; no. Suppl; p. S20
• Main Authors: Stoychev, Stoyan, Bekker-Jensen, Dorte, Martinez del Val, Ana, Steigerwald, Sophia, Batth, Tanveer, Gerber, Isak, Jordaan, Justin, Olsen, Jesper, Chien, Allis, Knudtson, Kevin, Martin, Roy
• Format: Journal Article
• Language: English
• Published: Bethesda, MD, USA Association of Biomolecular Resource Facilities 01.08.2020
• Subjects: Poster Abstracts
• ISSN: 1524-0215; 1943-4731

This work focusses on the application of high throughput workflows for proteome and phosphoproteome profiling followed by fast gradient liquid chromatography (LC) coupled to data-independent acquisition (DIA) mass spectrometry analysis. Automation of sample preparation increased throughput and reproducibility covering all steps from protein extract to mass spectrometry analysis allowing for parallel processing of up-to 96 samples in less than 6 hours (excluding digest time). We show the adaptation of the sample preparation methods including protein capture, clean-up, and on-bead digestion as well as phosphopeptide enrichment for use in a magnetic handling stations (Thermo Scientific King Fisher Flex) allowing for semi-automated sample processing. The methods are readily transferable to a range of liquid handling robots with magnetic handling stations (capabilities). Efficient protein isolation from wide range of tissues was achieved using 5% SDS followed by aggregation-based protein capture method (PAC) on MagReSyn® Amine magnetic microparticles allowing for efficient contaminant removal and on-bead digestion. For phosphoproteome profiling the PAC-generated peptides were further processed using MagReSyn® Ti-IMAC HP magnetic microparticles. All steps except for plate aliquoting and desalting were performed using a magnetic handling station that allowed for automated processing of up-to 96 samples in parallel. 500 ng peptides or enriched phosphopeptides were loaded directly on Evotips and further analysed using 21-minute gradients on Evosep LC coupled to Orbitrap Exploris equipped with FAIMS source allowing for up-to 60 samples per day to be analysed. Over 8,000 proteins and 22,000 unique phospho-peptides were quantified across the 12 tissues in a total mass spectrometry time of 28 hours.