title: Enhanced detection of in-gel released N-glycans by MALDI-TOF-MS.
abstract: Many biologically relevant glycoproteins need to be separated on 1D- or 2D-gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in-gel digestions with MALDI-TOF-MS. In this technical report, we investigated how the detection of in-gel released N-glycans could be improved by MALDI-TOF-MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D-arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl-β-glucopyranoside, a nonionic detergent, was studied during in-gel peptide-N(4) -(acetyl-ß-glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl-β-glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N-glycans. A LOD of under 7 pmol glycoprotein could be achieved.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry, and Pathobiochemistry, Charité Medical University, Berlin, Germany; Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Weiz
authors.forename: Aryaline
authors.initials: A
authors.lastname: Kamalakumar
authors.forename: Karina
authors.initials: K
authors.lastname: Biskup
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
mesh_terms: D000818:Animals; D002417:Cattle; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D003902:Detergents; D005960:Glucosides; D006023:Glycoproteins; D011134:Polysaccharides; C018619:octyl-beta-D-glucoside
keywords: Detergent; Glycan; Glycoproteomics; In-gel digestion; MALDI-TOF