Stoyan Stoychev, Ph.D., Stoyan has a background in Structural Biology and experience in the field of biological mass spectrometry having worked at a core facility for over 15 years. He currently holds a joint position at Evosep and ReSyn Biosciences with main focus on developing end-to-end (sample to to mass spectrometer) solutions for routine proteome profiling.
The robust quantitative characterization of proteins from plasma is critical to the diagnosis of disease and therapeutic monitoring. Membrane-bound particles in plasma are composed of extracellular vesicles, exosomes, and apoptotic bodies and represent ~1-2% of the total protein composition. Analysis of this enriched membrane particle fraction by mass spectrometry is effectively a “liquid biopsy,” and significantly improves the dynamic range of the proteins measurable in plasma. We have developed a one-step enrichment strategy (Mag-Net) using strong-anion exchange magnetic microparticles (ReSyn Biosciences) to capture membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, hemolysis compatible, inexpensive, and requires <100 μL plasma input. Coupled to a quantitative liquid chromatography mass spectrometry strategy using data independent acquisition, we demonstrate that we can routinely collect results for >42,000 peptides from >4,500 plasma proteins with high precision.
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In this presentation, we will illustrate how we used Skyline’s visualization capabilities to evaluate the data quality. This includes assessing the enrichment of known protein markers of exosomes and microvesicles, including CD9, CD61, ALIX, NCAM1, and flotillin-1. Importantly, >400 proteins were depleted relative to unfractionated plasma (e.g. albumin, transferrin, alpha-2-macroglobulin, alpha-1-antitrypsin, ApoA1, and ApoB). Using the Skyline external tool, Protter, we could rapidly evaluate whether proteins were transmembrane, lipid anchored, or peripheral membrane proteins – which totaled ~40% of the proteins measured. Skyline was used to also assess the quantitative precision of the sample preparation and the linearity and lower limit of quantitation (LLOQ) using a matrix matched calibration curve. Finally, Skyline and a shared folder on https://panoramaweb.org were used to share data and to evaluate assay performance between laboratories and instrument platforms.
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