Simultaneous Enrichment of Plasma Soluble and Extracellular Vesicular Glycoproteins Using Prolonged Ultracentrifugation-Electrostatic Repulsion-hydrophilic Interaction Chromatography (PUC-ERLIC) Approach*

Abstract

Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. Although intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinically useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation with electrostatic repulsion-hydrophilic interaction chromatography, to facilitate the recovery of both glycoproteins and extracellular vesicles from nondepleted human plasma. Following prolonged ultracentrifugation, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR < 1%), including 48 glycoproteins with concentrations ranging from pg to ng/ml. The novel enrichment method we report should facilitate future human plasmabased proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle-derived biomarkers, that can be used to predict clinical outcomes in human patients. Molecular & Cellular

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

“…ERLIC with volatile solvents has been successfully used in different proteomic experiments for protein identification, phosphorylation analysis and N-glycosylation studies [45–50]. We demonstrated here that the volatile formic acid/acetonitrile/water solvent system is also suitable for O-glycopeptide analysis, and eliminating a desalting step is definitely beneficial for the process.…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…Moreover, specific separation methods for the study of post-translational modifications of EV proteins have also been applied in many proteomic studies. As an example, the prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) was applied to study EV-enriched glycoproteins [ 128 ], while the immobilized metal ion affinity chromatography (IMAC) was employed to enrich phosphopeptides derived from the EV proteins’ digestion [ 129 ]. A few years ago, Wu et al also described a rapid isolation method known as EVTRAP for the highly efficient purification of EVs and further EV phosphoproteome analysis using polyMAC-based phosphopeptide enrichment [ 130 ].…”

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

“…Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for label-free proteomic analysis as described [ 42 ]. Briefly, LC-MS/MS (Agilent Technologies Inc., Santa Clara, CA, USA) was used to analyze purified proteins from the (V)LDL and HDL precipitates.…”