title: Characterization of sialylated and fucosylated glycopeptides of beta2-glycoprotein I by a combination of HILIC LC and MALDI MS/MS.
abstract: Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N-glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human beta2-glycoprotein I (beta2-GPI, apolipoprotein H), which contains multiple N-glycosylation sites. Conditions for rapid proteolytic digestion of beta2-GPI using low-specificity proteases were optimized to detect beta2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of beta2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Akira
authors.initials: A
authors.lastname: Kondo
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.forename: Karin
authors.initials: K
authors.lastname: Hjernø
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D005643:Fucose / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D053482:beta 2-Glycoprotein I / Q000737:chemistry*
chemical_list: D006020:Glycopeptides; D053482:beta 2-Glycoprotein I; D005643:Fucose; D019158:N-Acetylneuraminic Acid