Defining the\nSoluble and Extracellular Vesicle Protein\nCompartments of Plasma Using In-Depth Mass Spectrometry-Based Proteomics

Plasma-derived extracellular\nvesicles (pEVs) are a potential\nsource\nof diseased biomarker proteins. However, characterizing the pEV proteome\nis challenging due to its relatively low abundance and difficulties\nin enrichment. This study presents a streamlined workflow to identify\nEV proteins from cancer patient plasma using minimal sample input.\nStarting with 400 μL of plasma, we generated a comprehensive\npEV proteome using size exclusion chromatography (SEC) combined with\nHiRIEF prefractionation-based mass spectrometry (MS). First, we compared\nthe performance of HiRIEF and long gradient MS workflows using control\npEVs, quantifying 2076 proteins with HiRIEF. In a proof-of-concept\nstudy, we applied SEC–HiRIEF–MS to a small cohort (12)\nof metastatic lung adenocarcinoma (LUAD) and malignant melanoma (MM)\npatients. We also analyzed plasma samples from the same patients to\nstudy the relationship between plasma and pEV proteomes. We identified\nand quantified 1583 proteins in cancer pEVs and 1468 proteins in plasma\nacross all samples. While there was substantial overlap, the pEV proteome\nincluded several unique EV markers and cancer-related proteins. Differential\nanalysis revealed 30 DEPs in LUAD vs the MM group, highlighting the\npotential of pEVs as biomarkers. This work demonstrates the utility\nof a prefractionation-based MS for comprehensive pEV proteomics and\nEV biomarker discovery. Data are available via ProteomeXchange with\nthe identifiers PXD039338 and PXD038528.