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eSymposia | Proteomics in Cell Biology and Disease


A robust preparation method for micro-sample scale discovery proteomics


Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Description

A robust preparation method for micro-sample scale discovery proteomics Benjamin Cooper1,2,3*, Dalia Ponce1, Frédéric Hollande2,3, Jonathan Mangum1. 1. Department of Pharmacology & Therapeutics, University of Melbourne, Australia. 2. Department of Clinical Pathology, University of Melbourne, Australia 3. University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, Melbourne, Australia. The application of discovery proteomics to scarce samples is restricted by consumptive preparation methodologies. Here, we aimed to develop a sample preparation method which demonstrates robust, scalable, and affordable performance at the middle to low microgram scale. Using a variation of an in-StageTip (iST) protocol1, 50 µg of HEK-293T cell lysates were prepared with varying digestive enzyme concentrations, incubation times, and liquid strong cation exchange resin (SCX) volumes. Each variable condition was sequentially tested on three independent cell lysates with 10 µg of protein being analysed (LC-MS/MS with LFQ) using a 6545XT Advance-Bio ESI-LC-Q/TOF (Agilent Technologies). We observed the greatest number of IDs from protein samples incubated overnight with 1:50 Trypsin-LysC followed by peptide capture and sample clean-up using 10 µL of liquid SCX resin (mean number of IDs ± SD, 595 ± 18). We sought to benchmark this performance of our ‘homemade’ liquid SCX inStage-Tip (HiST) against a commercially available iST sample preparation kit (PreOmics). Our method generated significantly greater numbers of protein IDs (mean number of IDs ± SD, 376 ± 95) and lower levels of intra-replicate variability (CV = HiST 3.08 vs. PreOmics 25.25) when compared with the commercial kit. These data highlight the robustness of our method in middle to low microgram sample preparation contexts and supports its use in future high-throughput proteomic analysis of scarce samples. 1 Kulak, N., Pichler, G., Paron, I. et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319–324 (2014). https://doi.org/10.1038/nmeth.... This research was supported by Agilent technologies in the provision of MS equipment and PhD stipend for lead Author. This research is also supported by an Australian Government Research Training Program (RTP) Scholarship. *email benc1@student.unimelb.edu.au

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