on 10-30-201806:56 AM - edited on 10-15-202110:54 AM by Closed Account
Sergei Snovida; Katherine Herting; Ramesh Ganapathy; Barbara Kaboord; John Rogers
Purpose: Depletion of abundant plasma proteins is required to identify and measure changes in prognostic or diagnostic proteins. We have developed new top2 and top14 abundant protein depletion resins and evaluated them for depletion specificity, efficiency, and reproducibility. We also combined our abundant protein depletion resins with a streamlined Tandem Mass Tag (TMT) sample preparation procedure.
Methods: Commercially-obtained pooled human plasma samples were used to assess depletion resins by SDS gel, ELSIA, and LC-MS. High pH reversed-phase fractionation was performed on TMT-labeled samples before LC-MS analysis. All samples were analyzed on Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer and processed using Thermo Scientific Proteome Discoverer 2.2 software.
Results: After optimization of the antibody ligand conjugation chemistries and resin blending protocols, we achieved >97-99% depletion efficiency of twelve high abundance protein targets in human plasma samples. Abundant protein depletion from plasma enabled detection of 34% more peptides and 25% more proteins compared to undepleted samples. Assessment of depletion capacity of the new resins showed high reproducibility for samples ranging from 10- 100ul. These depletion resins were also used in combination with a new sample preparation workflow for TMT-reagent labeling which minimizes sample handling and the number of steps. Using this workflow, we identified and quantified nearly 1100 proteins in normal and lung cancer patient plasma samples.
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