panoramaProteomicsMichael MacCossHomo Sapienshttps://panoramaweb.org/Mag-Net.urlmaccoss@uw.eduUniversity of WashingtonPanoramaPublicExtracellular vesicles (EVs) in plasma are composed of exosomes, microvesicles, and apoptotic bodies. We report a plasma EV enrichment strategy using magnetic beads called Mag-Net. Proteomic interrogation of this plasma EV fraction enables the detection of proteins that are beyond the dynamic range of liquid chromatography-mass spectrometry of unfractionated plasma. Mag-Net is robust, reproducible, inexpensive, and requires <100 μL plasma input. Coupled to data-independent mass spectrometry, we demonstrate the measurement of >37,000 peptides from >4,000 proteins. Using Mag-Net on a pilot cohort of patients with neurodegenerative disease and healthy controls, we find 204 proteins that differentiate (q-value < 0.05) patients with Alzheimer's disease dementia (ADD) from those without ADD. There are also 310 proteins that differ between individuals with Parkinson's disease and without. Using machine learning we distinguish between individuals with ADD and not ADD with an area under the receiver operating characteristic curve (AUROC) = 0.98 ± 0.06.Enrichment of extracellular vesicles using Mag-Net for the analysis of the plasma proteome.Wu Christine C CC, Tsantilas Kristine A KA, Park Jea J, Plubell Deanna D, Sanders Justin A JA, Naicker Previn P, Govender Ireshyn I, Buthelezi Sindisiwe S, Stoychev Stoyan S, Jordaan Justin J, Merrihew Gennifer G, Huang Eric E, Parker Edward D ED, Riffle Michael M, Hoofnagle Andrew N AN, Noble William S WS, Poston Kathleen L KL, Montine Thomas J TJ, MacCoss Michael J MJAW488255, Fresh, DmelCG5932, Elk, ELK, C130099E04Rik, Frozen Plasma, Exovesicles, Fresh Frozen Plasma, determination, Apoptotic Body, Bodies, Neuronally-expressed EPH-related tyrosine kinase, Blood, Apoptotic Bodies, NetrinA, EPH tyrosine kinase 2, Vesicles, D430049E23Rik, total expressed protein, SAP2, Extracellular Vesicle, 9330129L11, neuroendocrine tumor, Gma, GMA, Plasmas, net, SIGLEC-4A, S-MAG, neuroendocrine neoplasm, Vesicle, netrin, siglec-4a, Frozen Plasmas, Hek6, Norepinephrine transporter, Apoptotic, chemical analysis, Proteomes., CG5932, Cek6, EPH-like kinase 6, Exovesicle, ENSMUSG00000074119, Plasma, ERP, APUDoma, Erp, Fresh Frozen Plasmas, Fresh Frozen, Blood Plasma, Elkh, Tyrosine-protein kinase receptor EPH-2, EK6, portion of plasma, SAP-2, hEK6, Sap-2, CG18657, Body, neuroendocrine tumour, DmelCG18657, 2.7.10.1, Solute carrier family 6 member 2, EPHT2, Etrp, Extracellular, Blood Plasmas, CT27014, NET1, SLC6A5, NAT1, assay, netA, SIGLEC4A, netrin A, NET, Net, plasmaAW488255, Fresh, Membrane Tissue, membrane, DmelCG5932, Elk, ELK, C130099E04Rik, Frozen Plasma, Fresh Frozen Plasma, determination, Neuronally-expressed EPH-related tyrosine kinase, Blood, NetrinA, EPH tyrosine kinase 2, number, D430049E23Rik, integral to membrane, total expressed protein, SAP2, 9330129L11, neuroendocrine tumor, Gma, GMA, Plasmas, net, presence, SIGLEC-4A, S-MAG, neuroendocrine neoplasm, netrin, siglec-4a, Frozen Plasmas, Membrane Tissues, count in organism, Hek6, Norepinephrine transporter, integral component of membrane, chemical analysis, Proteomes., whole membrane, CG5932, Cek6, EPH-like kinase 6, ENSMUSG00000074119, Plasma, ERP, APUDoma, Erp, Fresh Frozen Plasmas, Fresh Frozen, Blood Plasma, Elkh, transmembrane, Tissues, membrane region, Tyrosine-protein kinase receptor EPH-2, EK6, Tissue, portion of plasma, SAP-2, hEK6, Sap-2, Membrane, CG18657, neuroendocrine tumour, DmelCG18657, 2.7.10.1, Solute carrier family 6 member 2, EPHT2, Etrp, Blood Plasmas, CT27014, NET1, SLC6A5, region of membrane, NAT1, assay, netA, SIGLEC4A, netrin A, NET, Net, plasmaPilot, Primary senile degenerative dementia, Presenile Alzheimer Dementia, Parkinson's disease, AU023367, Early Onset, Parkinson's disease NOS, Amentias, Blood, NetrinA, Liquid Chromatography Mass Spectrometry, PILOT, D430049E23Rik, High Performance Liquid Chromatography Mass Spectrometry, Neurologic Diseases, Extracellular Vesicle, Progress Reports, Ultra Performance Liquid Chromatography-Mass Spectrometry, Neurologic Degenerative Condition, Gma, GMA, Early Onset Alzheimer Disease, "Neurodegenerative disease" EXACT [NCI2004_11_17:C4802], Alzheimer's disease, S-MAG, High Performance Liquid Chromatography-Mass Spectrometry, 1B1, Primary Senile Degenerative, Polypeptides, AD, anon-EST:Posey9, HPLC-MS, Liquid Chromatography-Mass, peptido, Paralysis Agitans, Neurologic Disorders, Summary Report, CG5932, Analysis, dementia (disease), Transfer, dementia, Summary Reports, Paranoid Dementia, ROC Analysis, Mass Spectrum Analysis, Nervous System Degenerative Diseases, Fresh Frozen Plasmas, Fresh Frozen, Ultra Performance Liquid Chromatography Mass Spectrometry, Elkh, Degenerative Condition, Familial Alzheimer Disease (FAD), Alzheimer Diseases, Parkinson disease, peptides, Progress Report, Analyses, Alzheimer syndrome, Parkinson's disease (disorder), Machine, Amentia, 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Attention Deficit Hyperactivity Disorder, Summary, EST D, Familial Alzheimer Diseases (FAD), netrin A, Field Reportliquid chromatography tandem mass spectroscopy, Fresh, AW488255, GRP1/cytohesin 1, Fresh Frozen Plasma, Procedures, Blood, NetrinA, number, D430049E23Rik, Gene, Spectrum Analyses, Gma, GMA, composed of, presence, S-MAG, LC-MS-MS, netrin, CG11633, cytohesin/GRP1, Membrane Tissues, Polypeptides, method, Techniques, peptido, resilient, Hek6, Method, integral component of membrane, GRP1, tough, method used in an experiment, LC-MSMS, Grp1, Gene Products, Studies, Mass, CG5932, anion, Cek6, cytopathology, Analysis, Technique, Mass Spectroscopy, ENSMUSG00000074119, Mass Spectrum Analysis, ERP, APUDoma, Erp, strong, Fresh Frozen Plasmas, Fresh Frozen, Blood Plasma, aniones, Elkh, LCMSMS, Biopsies, peptides, Analyses, Tissues, PTPSTEP, membrane region, Tissue, Tyrosine-protein kinase receptor EPH-2, EK6, l(2)SH2 0323, composition, SAP-2, Sap-2, Anion, Study, neuroendocrine tumour, 2.7.10.1, Neural-specific protein-tyrosine phosphatase, Solute carrier family 6 member 2, Methodological Studies, method used in an experiment., l(2)k08110, Etrp, Blood Plasmas, CT27014, region of membrane, NET1, SLC6A5, peptidos, NAT1, netA, plasma, NET, Net, GPH, LC-MS2, Membrane Tissue, membrane, DmelCG5932, Elk, ELK, Frozen Plasma, C130099E04Rik, Neuronally-expressed EPH-related tyrosine kinase, Proteins, LC-MS/MS, EPH tyrosine kinase 2, integral to membrane, stepk, SAP2, 9330129L11, compositionality, neuroendocrine tumor, Procedure, Plasmas, net, Spectrum Analysis, SIGLEC-4A, results, Peptide, neuroendocrine neoplasm, Anionen, Spectroscopy, Frozen Plasmas, siglec-4a, count in organism, LC/MS/MS, MS, Norepinephrine transporter, Protein, 3.1.3.48, whole membrane, EPH-like kinase 6, simple tissue, l(2)SH0323, Mass Spectrum Analyses, CYH1, Plasma, Mass Spectrum, transmembrane, histopathology, Step, CG11628, content, Spectrometry, Magnetic, portion of plasma, Striatum-enriched protein-tyrosine phosphatase, Methodological, hEK6, Membrane, study protocol, CG18657, Methodological Study, anions, DmelCG11628, Protein Gene Products, plan specification, Gene Proteins, DmelCG18657, STEP, EPHT2, liquid chromatography-tandem mass spectroscopy, structure, liquid chromatography tandem mass spectrometry, Peptid, Polypeptide, biopsy, SIGLEC4A, netrin AfalseMag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteomeMembrane-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.Wed Jul 02 00:00:00 GMT+01:00 2025PXD042947960640595564