Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome
- Organism: Homo sapiens
- Instrument: Orbitrap Eclipse
extracellular vesicles, DIA mass spectrometry, plasma proteomics, quantitative proteomics, neurodegenerative disease
Lab head: Michael MacCoss
Submitter: Michael MacCoss
Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a “liquid biopsy,” while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry in unfractionated plasma. We have developed a one-step enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 μL plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can routinely collect results for >37,000 peptides from >4,000 plasma proteins with high precision. We demonstrate excellent quantitative accuracy and analytical reproducibility of the protocol.
Here we describe Mag-Net, a robust, inexpensive, and simple magnetic bead based method to capture EV particles from plasma while simultaneously depleting abundant plasma proteins. The method enriches EVs from plasma using a combination of size and charge. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate routine measurement of >37,000 peptides from >4,000 plasma proteins with high precision. Interestingly, 21.4% proteins measured by Mag-Net have a predicted transmembrane domain by either Philius or DeepTMHMM, with an additional 5-10% known to be lipid anchored proteins or peripherally associated membrane proteins, a category of proteins often under-represented in proteomic analyses, particularly from plasma. We characterize the proteins captured by Mag-Net, the physical characteristics of the captured membrane particles, and demonstrate the value of this method for high coverage quantitative analysis of the plasma proteome. We believe this easy to perform, cost effective solution will enable the identification of novel markers of disease and the corresponding response to therapy.
Plasma samples from 17 individual healthy donors collected in K2EDTA vials were purchased from Innovative Research (IPLASK2E2ML, Innovative Research Inc, Novi, MI).
Created on 6/13/23, 9:40 AM