Proteomic Analysis of Plasma – Sample Preparation and Multiplexing Workflows for Relative Quantitation

[Orbitrap_SciLib]

Sergei I. Snovida1; Yen-Chun Lai2, Amarjeet Flora1, Ryan Bomgarden1, John. C. Rogers1
ASMS 2019 Poster
Purpose: Depletion of abundant proteins is required to identify and measure changes in prognostic or diagnostic plasma proteins. We have implemented the use of top14 abundant protein depletion resin, efficient sample preparation using Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit, labeling peptide-level samples with Tandem Mass Tag™ (TMT™) reagents, and high pH reversed-phase fractionation to profile a large set of plasma samples from normal controls and patients with pulmonary hypertension heart failure with preserved ejection fraction (PH-HFpEF) to assess differences in plasma protein abundances. Methods: Plasma samples were subjected to top14 abundant protein depletion, followed by a quick sample preparation using the EasyPep Mini sample preparation kit. Samples were then grouped into five 11-plex sets and labeled with TMT-reagents. Following the labeling, each grouped set was fractionated by high pH reversed-phase into eight fractions. Fractions were analyzed by LC-MS on Thermo Scientific™ Orbitrap Fusion™ instrument using synchronous precursor selection mode (SPS) for accurate, interference-free quantitative comparison on a 75 cm C18 column in a 3 hour gradient. Raw files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software. Results: We have designed a study in which thirty-two plasma samples from healthy donors and individuals diagnosed with pulmonary hypertension heart failure with preserved ejection fraction (PH HFpEF) were prepared and analyzed using the above workflow in five 11-plexed sets, each set consisting of pooled controls and bridging channels. Protein abundances were compared across all individuals as well as between the two pooled cohorts to identify potential biomarker candidates and gain insight into the mechanism of the development and progression of the PH-HFpEF. Overall, 1043 protein groups and 8580 peptide groups were identified in this experiment. Several proteins were shown to have differential abundance in the PH-HFpEF patients samples relative to the normal controls.


1. Thermo Fisher Scientific, Rockford, IL, USA 2. Indiana University School of Medicine, IN, USA