title: Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.
abstract: Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.
authors: Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
authors: Shin-Yi
authors: SY
authors: Yu
authors: Sz-Wei
authors: SW
authors: Wu
authors: He-Hsuan
authors: HH
authors: Hsiao
authors: Kay-Hooi
authors: KH
authors: Khoo
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D054794:Glycomics / Q000379:methods*; D006801:Humans; D008198:Lymph Nodes / Q000737:chemistry; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011134:Polysaccharides; D013431:Sulfates