Simultaneous Enrichment of Plasma Extracellular Vesicles and Glycoproteome for Studying Disease Biomarkers.

To detect disease at an early stage and to develop effective disease treatment therapies, reliable biomarkers of diagnosis, disease progression, and its status remain a research priority. A majority of disease pathologies are primarily associated with different subsets of cells of different tissues, discrete compartments, and areas. These subsets of cells release glycoproteins and specific extracellular vesicles (EVs) including microvesicles and exosomes that carry bioactive cargoes of proteins, nucleic acids, and metabolites. Body fluids like blood plasma are considered as a golden source of disease biomarkers since it contains glycoprotein and EVs released by almost all cell types. The contents of glycoproteome and EV cargo change with cell status, and they act as mirror of cell's intracellular events and status; hence, EVs and glycoproteins are promising disease biomarkers. However, their abundance in blood plasma remains low posing a serious technical problem in their identification and quantification. Until recently, technical advances and exhaustive research devised a technique for either enrichment of plasma glycoprotein or EVs, but no methodologies exist that can enrich and identify both plasma glycoprotein and EVs. To overcome this technical challenge, a method that can eliminate high-abundance entities without depleting disease-modifying molecules is required. Therefore, here we describe the detailed protocol of simultaneous enrichment of glycoproteins and EVs from blood plasma by prolonged ultracentrifugation coupled to electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) and their identification and quantification by mass spectrometry-based proteomic technique.