title: A sensitive gel-based global O-glycomics approach reveals high levels of mannosyl glycans in the high mass region of the mouse brain proteome.
abstract: We developed a gel-based global O-glycomics method applicable for highly complex protein mixtures entrapped in discontinuous gradient gel layers. The protocol is based on in-gel proteolysis with pronase followed by (glyco)peptide elution and off-gel reductive β-elimination. The protocol offers robust performance with sensitivity in the low picomolar range, is compatible with gel-based proteomics, and shows superior performance in global applications in comparison with workflows eliminating glycans in-gel or from electroblotted glycoproteins. By applying this method, we analyzed the O-glycome of human myoblasts and of the mouse brain O-glycoproteome. After semipreparative separation of mouse brain proteins by one-dimensional SDS gel electrophoresis, the O-glycans from proteins in different mass ranges were characterized with a focus on O-mannose-based glycans. The relative proportion of the latter, which generally represent a rare modification, increases to comparatively high levels in the mouse brain proteome in dependence of increasing protein masses.
authors.affiliation: Medical Faculty, Institute of Biochemistry II, University of Cologne, Joseph-Stelzmann-Str., 50931 Cologne, Germany. Isabelle.Breloy@uni-koeln.de
authors.forename: Isabelle
authors.initials: I
authors.lastname: Breloy
authors.forename: Sandra
authors.initials: S
authors.lastname: Pacharra
authors.forename: Christina
authors.initials: C
authors.lastname: Aust
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002240:Carbohydrate Sequence; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D008358:Mannose / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis*; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D020543:Proteome; D008358:Mannose