title: 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 plasma-based 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.
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551;
authors: Esther
authors: E
authors: Sok Hwee Cheow
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551;
authors: Kae
authors: K
authors: Hwan Sim
authors: §Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, and Cardiovascular Research Institute, Singapore 119228; ¶Experimental Cardiology Laboratory, Cardiology, University Medical Center Utrecht, the Netherlands & Interuniversity Cardiovascular Institute of the Netherlands, Utrecht, the Netherlands;
authors: Dominique
authors: D
authors: de Kleijn
authors: §Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, and Cardiovascular Research Institute, Singapore 119228; ‖National University Heart Centre, Department of Cardiac, Thoracic and Vascular Surgery, Singapore 119228; **Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228.
authors: Chuen
authors: C
authors: Neng Lee
authors: §Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, and Cardiovascular Research Institute, Singapore 119228; ‖National University Heart Centre, Department of Cardiac, Thoracic and Vascular Surgery, Singapore 119228; **Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228.
authors: Vitaly
authors: V
authors: Sorokin
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551; sksze@ntu.edu.sg.
authors: Siu Kwan
authors: SK
authors: Sze
mesh_terms: D002845:Chromatography / Q000379:methods; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006331:Heart Diseases / Q000097:blood; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010949:Plasma / Q000737:chemistry; D020543:Proteome; D055672:Static Electricity; D014461:Ultracentrifugation
chemical_list: D006023:Glycoproteins; D020543:Proteome