Defining the Soluble and Extracellular Vesicle Protein Compartments of Plasma Using In-Depth Mass Spectrometry-Based Proteomics

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