title: Boric acid gel enrichment of glycosylated proteins in human wound fluids.
abstract: The enrichment of glycosylated proteins by glycocapturing materials plays a pivotal role for the investigation of polysaccharide containing proteins in disease pathogenesis. Hence, we investigated a boric acid gel as a binding material for glycoprotein enrichment. The bovine proteins alpha-1-acid-glycoprotein (A1AG) and alpha-2-HS-glycoprotein (fetuin A) were spiked in human chronic wound fluids and were subsequently enriched by a boric acid gel affinity chromatography (BAGAC). The enrichment efficiency was evaluated by western blot analysis and mass spectrometry. Additionally, glycoproteins of human wound fluids from diabetes mellitus patients with chronic foot ulcers were analyzed after BAGAC enrichments. In total 104 glycoproteins were identified, with reported glycosylation sites. 60 proteins were detected in at least 2 out of 3 biological replicates and were used for quantitative analysis between the bound and unbound fractions. Almost 80% of these glycoproteins were more prominent in the bound fraction. Only 2 glycoproteins revealed higher spectral counts in the flow through fraction compared to the bound fraction. These findings demonstrate the capability of the BAGAC material to enrich glycosylated proteins from complex human wound fluids.
authors.affiliation: Department of Analytical Chemistry, Ruhr-University Bochum, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Krisp
authors.forename: Caroline
authors.initials: C
authors.lastname: Kubutat
authors.forename: Andreas
authors.initials: A
authors.lastname: Kyas
authors.forename: Lars
authors.initials: L
authors.lastname: Steinsträsser
authors.forename: Frank
authors.initials: F
authors.lastname: Jacobsen
authors.forename: Dirk
authors.initials: D
authors.lastname: Wolters
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001826:Body Fluids / Q000737:chemistry* / Q000378:metabolism; D001888:Boric Acids / Q000494:pharmacology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D005782:Gels / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D054928:Validation Studies as Topic; D014947:Wounds and Injuries / Q000378:metabolism*
chemical_list: D001888:Boric Acids; D005782:Gels; D006023:Glycoproteins; C032688:boric acid