Development and Applications of Mass Spectrometry-based Sulfoglycomics
Date Issued
2011
Date
2011
Author(s)
Yu, Shin-Yi
Abstract
Sulfate modification on terminal oligosaccharyl epitopes carried on N-glycans and O-glycans has increasingly been indicated as critical determinant mediating a diverse range of biological functions. For example, lymphocyte homing to lymphoid tissue is critically dependent on recognition between L-selectin on lymphocytes and 6-sulfo sLeX epitope carried on the O-glycans on the high endothelial venule of peripheral lymph nodes. Additionally, many other sulfated glycotopes have recently been identified as preferred ligands for Siglecs and galectins in immune systems via glycan array screening. To define its true physiological relevance, a robust sulfoglycomic analysis is needed to establish the expression pattern, exact structures and the glycan carriers of these sulfated epitopes.
Due to a natural low abundance and the negatively charged nature, sulfated glycans are often not easily detected by current approaches in mass spectrometry (MS) based glycomic analysis. This thesis work aims to develop methodology for sulfoglycomic analysis, and to apply the developed methodology to different biological samples. First, the standard permethylation protocol was further developed and optimized for sulfated glycans, allowing efficient recovery of the permethyl derivatives by use of reverse phase C18 cartridge for a high sensitivity first screen by MALDI-MS in the negative ion mode. A better matrix, 3,4-diaminobenzophenone, was identified, which significantly enhances detection sensitivity. Moreover, through systematic MALDI-MS/MS analysis of synthetic sulfated glycan standards in negative ion mode, we identified diagnostic fragment ions to unambiguously determine the location and linkage of the sulfate moiety. Subsequent microscale fractionation based on NH2 beads packed in microtip offers enrichment of sulfated glycans away from highly abundant non-sulfated glycans, so as to enable better chance of detection of sulfated glycans in the positive ion mode. It was found that previously established low and high energy CID MS/MS fragmentation characteristics in the positive ion mode are directly applicable to permethylated sulfated glycans, which enable a complete sequencing including glycosyl linkage and branching determination that are otherwise less amenable in negative ion mode.
The developed methodology provided a high sensitivity mapping of the sulfoglycomes derived from mouse lymph nodes and human GlcNAc-6-sulfotransferase transfected cell lines. It was found that 6-sulfo sLeX glycotope can be carried on N-glycans and not restricted in expression to O-glycans of mouse peripheral lymph nodes. Moreover, sulfated N-glycans and O-glycans were still detected in mice with double knockouts of the critical GlcNAc6ST-1 and -2, suggesting the contribution of other sulfotransferases in making the residual sulfated epitopes including those sulfated at Gal. Distinctive sulfoglycomic pattern of mouse Peyer’s patches was observed in comparison with that of peripheral lymph nodes, and a novel sulfated SdA-like glycotope carried on O-glycans was identified by MS/MS sequencing and further linkage analysis. Finally, in the case of colon cancer cells transfected with different human GlcNAc6STs, it was found that only human GlcNAc6ST-2 can add sulfate on both core 2 and extended core 1 based O-glycans, whereas both GlcNAc6ST-1 and GlcNAc6ST-3 will not act on extended core 1 structures. These results are fully consistent with elevated expression of GlcNAc6ST-2 in colonic carcinomas, which contributed to positive MECA79 immunostaining against the sulfated glycotope carried on extended Core 1 structures.
Subjects
Sulfoglycomics
mass spectrometry
permethylation
glycan sequencing
SDGs
Type
thesis
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