Authors: PingFu Cheng Sergei Snovida MingYi Ho ChuWen Cheng Albert M Wu KayHooi Khoo
Publish Date: 2013/06/25
Volume: 405, Issue: 21, Pages: 6683-6695
Abstract
Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4GlcNAccapped terminal epitope functionally implicated in protection against Helicobacter pylori infection Being more readily available and reasonably well characterized it serves as a good reagent for immunobiological studies as well as a standard for analytical methodology developments Current approaches in mass spectrometry MSbased glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation particularly for cases such as the extremely heterogeneous Oglycosylation of mucosal mucins that can be further sulfated We demonstrate here a novel concerted workflow that extends the conventional matrixassisted laser desorption/ionization–mass spectrometry MALDIMS mapping of permethylated glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode This was facilitated by introducing a mixedmode solidphase extraction step which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate nonsulfated and sulfated pools in one single step By distinct MALDIMS/MS fragmentation patterns all previously known structural features of porcine gastric mucin including the terminal epitopes and location of sulfates could be readily defined We additionally showed that both arms of the core 2 structures could be extended via 6Osulfated GlcNAc to yield a series of disulfated Oglycans not previously reported thus expanding its current glycomic coverage However a targeted LCMSn analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y epitope implicated by positive antibody bindingThis work was supported by Academia Sinica and Taiwan National Science Council NSC grant 992311B001021MY3 to KKH NSC grant 1012320B182011 and ChangGung Medical Research Project CMRP grant nos 180482 and 170443 to WAM The MS data were acquired at the previous NRPGM Core Facilities for Proteomics and Glycomics NSC 993112B001025 and current Core Facilities for Protein Structural Analysis at Academia Sinica supported under the Taiwan National Core Facility Program for Biotechnology NSC 1002325B001029 NSC1012319B001003 The authors thank Ms YuPing Gong for technical assistance and data collection
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