title: Open-tubular capillary electrochromatography with bare gold nanoparticles-based stationary phase applied to separation of trypsin digested native and glycated proteins.
abstract: In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. miksik@biomed.cas.cz
authors.forename: Ivan
authors.initials: I
authors.lastname: Mikšík
authors.forename: Kateřina
authors.initials: K
authors.lastname: Lacinová
authors.forename: Zdeňka
authors.initials: Z
authors.lastname: Zmatlíková
authors.forename: Pavla
authors.initials: P
authors.lastname: Sedláková
authors.forename: Vladimír
authors.initials: V
authors.lastname: Král
authors.forename: David
authors.initials: D
authors.lastname: Sýkora
authors.forename: Pavel
authors.initials: P
authors.lastname: Rezanka
authors.forename: Václav
authors.initials: V
authors.lastname: Kašička
mesh_terms: D000327:Adsorption; D000818:Animals; D053801:Capillary Electrochromatography / Q000295:instrumentation / Q000379:methods*; D006031:Glycosylation; D006046:Gold / Q000737:chemistry; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010455:Peptides; D011506:Proteins; D006046:Gold; D014357:Trypsin