ECD AND EDD OF NATIVE AND PERMETHYLATED GLYCANS

天然和全甲基化聚糖的 ECD 和 EDD

• 批准号: 7955963
• 负责人: PETER B. O'CONNOR
• 金额: $ 2.83万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955963
• 关键词: Biology; Charge; Complex; Computer Retrieval of Information on Scientific Projects Database; Custom; Fourier transform ion cyclotron resonance; Funding; Glycoproteins; Grant; Institution; Ions; Isomerism; Link; Mannose; Manuscripts; Mass Spectrum Analysis; Medicine; Molecular; Oligosaccharides; Pattern; Polysaccharides; Positioning Attribute; Protocols documentation; Publishing; Research; Research Personnel; Resources; SHFM1 gene; Source; Structure; United States National Institutes of Health; Vertebral column; improved; maltoheptaose; research study; sodium cyanoborohydride

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CAD, "hot" ECD and EDD have been utilized to study the fragmentation patterns of linear and branched glycans. Sodiated and permethylated glycans were analyzed by both CAD and "hot" ECD experiments available in a custom-built ESI-FTICR MS, and the resulting spectra provided complementary structural information. CAD generated major B and Y fragment ions whereas while C and Z products dominated the "hot" ECD produced major C and Z ions. A-type cross-ring cleavages were present in spectra generated by CAD while complementary A- and X-type pairs happened resulted from "hot" ECD. Especially 0,4An and 3,5An ions defined the linkage position of the upper branch. More abundant internal fragments were observed in CAD than in "hot" ECD spectra. Since acidic glycans form more stable spray in the negative mode than in the positive mode, the EDD experiment on the native glycans was performed in the negative mode; it generated more cross-ring cleavages than CAD. The native glycans, including those released from glycoproteins, were permethylated using our standard protocol. The results are summarized briefly here. Sodiated and permethylated linear malto-oligosaccharides: The fragmentation patterns of sodiated and permethylated linear (Glc)6-(Glc)9 are very similar. The glycosidic cleavages (B, Y, C, and Z ions), cross-ring cleavages (A and X ions), and internal cleavages (B/Y and C/Y ions) were all observed. Due to their highly symmetric structures, Bn and Zn, Cn and Yn, 0,2Xn and 2,4An+1 ions are isobaric. In order to differentiate the pairs, the maltoheptaose was reduced by sodium cyanoborohydride. Extensive fragment ions were detected in CAD and the Y ions had the highest abundance. "Hot" ECD provided cleavages similar to CAD, though with fewer cross-ring cleavages. Sodiated and permethylated N-linked branched glycans: CAD and "hot" ECD on sodiated and permethylated high-mannose and complex type (asialo-, disialyated biantennary) N-linked glycans provided complementary structural information. The sequences of these glycans were confirmed by B, Y ions in CAD and C, Z ions in "hot" ECD. Branching, composition and linkage information was determined by cross-ring cleavages (A-type in CAD and complementary A and X pairs in "hot" ECD). Internal fragments are particularly frequent in CAD but also occur in ECD. Higher collision energy was required to fragment the backbone of sialylated glycans to the same extent as their asialo counterparts. The triply charged molecular ion of a sodiated and permethylated disialylated-biantennary N-linked glycan was abundant and was fragmented by the "hot" ECD generating extensive fragment ions (glycosidic and complementary pairs of cross-ring cleavages) to fully confirm its sequence, branching, and linkage assignments. Fragmentation of the larger high-mannose type glycans such as GlcNAc2Man7-9 by ECD is still difficult but may be improved by activated ion ECD. EDD of native linear and branched glycans: EDD experiments on the native linear and branched N-linked glycans generated more cross-ring cleavages than CAD; these cleavages could be used to differentiate isomers. The ECD manuscript was published, the EDD manuscript has been submitted and is currently under revision after the initial reviews were received.

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