Abstract
Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4-GlcNAc-capped 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 (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) 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 mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/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 6-O-sulfated GlcNAc to yield a series of disulfated O-glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MSn 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 binding.
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Abbreviations
- CID:
-
Collision-induced dissociation
- lacdiNAc:
-
N,N′-Diacetyllactosamine
- lacNAc:
-
N-Acetyllactosamine
- mAb:
-
Monoclonal antibody
- MS:
-
Mass spectrometry
- SPE:
-
Solid-phase extraction
- TFA:
-
Trifluoroacetic acid
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Acknowledgments
This work was supported by Academia Sinica and Taiwan National Science Council (NSC) grant 99-2311-B-001-021-MY3 (to KKH), NSC grant 101-2320-B-182-011, and Chang-Gung 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 99-3112-B-001-025), and current Core Facilities for Protein Structural Analysis at Academia Sinica, supported under the Taiwan National Core Facility Program for Biotechnology, NSC 100-2325-B-001-029, NSC101-2319-B-001-003. The authors thank Ms. Yu-Ping Gong for technical assistance and data collection.
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Cheng, PF., Snovida, S., Ho, MY. et al. Increasing the depth of mass spectrometry-based glycomic coverage by additional dimensions of sulfoglycomics and target analysis of permethylated glycans. Anal Bioanal Chem 405, 6683–6695 (2013). https://doi.org/10.1007/s00216-013-7128-2
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DOI: https://doi.org/10.1007/s00216-013-7128-2