title: Improved in-gel reductive β-elimination for comprehensive O-linked and sulfo-glycomics by mass spectrometry.
abstract: Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia; Department of Biochemistry and Molecular Biology, University of Georgia.
authors.forename: David B
authors.initials: DB
authors.lastname: Nix
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia.
authors.forename: Tadahiro
authors.initials: T
authors.lastname: Kumagai
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia; Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia; Department of Biochemistry and Molecular Biology, University of Georgia.
authors.forename: Michael
authors.initials: M
authors.lastname: Tiemeyer
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia; kaoki@ccrc.uga.edu.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
mesh_terms: D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D054794:Glycomics / Q000379:methods; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009077:Mucins / Q000032:analysis; D011134:Polysaccharides / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012463:Saliva / Q000737:chemistry; D013431:Sulfates / Q000032:analysis
chemical_list: D002412:Cations; D006023:Glycoproteins; D009077:Mucins; D011134:Polysaccharides; D013431:Sulfates