on 06-12-201802:21 PM - edited on 10-15-202110:54 AM by Closed Account
Sergei Snovida; Katherine Herting; Ramesh Ganapathy; Ryan Bomgarden; Barbara Kaboord; Chris Etienne; John Rogers
ASMS 2018 Purpose: The large dynamic range in protein abundance of plasma samples is the main problem associated with plasma/serum-based biomarker discovery experiments, and depletion of abundant proteins is required to identify and measure changes in prognostic or diagnostic plasma proteins. We have optimized the production and immobilization of immunoaffinity ligands to develop new top2 and top12 abundant protein depletion resins. In addition, we comprehensively evaluated the specificity, efficiency, and reproducibility abundant protein depletion from human plasma samples. Finally, we demonstrated the use of these new depletion resins to identify differences in plasma from lung cancer patients using a multiplexed protein quantitation workflow.
Methods: Relative abundance of human plasma proteins was determined by ELISA and LC-MS. LC-MS samples were analyzed on Thermo Scientific Orbitrap FusionTM TribridTM mass spectrometer and processed using Thermo Scientific™ Proteome Discoverer™ 2.2 and Skyline 3.6 (University of Washington) software.
Results: After optimization of the antibody ligand conjugation chemistries and resin blending protocols, we achieved >97-99% depletion efficiency of high abundance protein targets in human plasma samples. Assessment of depletion capacity of the new resins show high depletion efficiencies, post-depletion protein yields, and reproducibility over a defined range of sample amounts. The utility of the procedure was further demonstrated using a TMT reagent-based quantitative proteomics experiment which identified and quantified nearly 1100 proteins in normal and lung cancer patient plasma samples.
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