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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 CAD、“热”ECD和EDD已被用于研究直链和支链聚糖的断裂模式。通过定制的ESI-FTICR MS中可用的CAD和“热”ECD实验对钠化和全甲基化聚糖进行了分析，所得光谱提供了补充结构信息。 CAD产生主要的B和Y碎片离子，而C和Z产物占主导地位，“热”ECD产生主要的C和Z离子。 CAD产生的谱图中存在A型交叉环裂解，而热ECD产生互补的A型和X型对。 特别是0，4 An和3，5 An离子确定了上分支的连接位置。 在CAD中观察到比在“热”ECD光谱中更丰富的内部片段。由于酸性聚糖在负模式下比在正模式下形成更稳定的喷雾，因此在负模式下对天然聚糖进行EDD实验;其产生的交叉环裂解比CAD更多。天然聚糖，包括从糖蛋白释放的聚糖，使用我们的标准方案进行全甲基化。结果简要总结如下。钠化和全甲基化线性麦芽低聚糖：钠化和全甲基化线性（Glc）6-（Glc）9的片段化模式非常相似。观察到糖苷裂解（B、Y、C和Z离子）、交叉环裂解（A和X离子）和内部裂解（B/Y和C/Y离子）。 由于它们的高度对称结构，Bn和Zn，Cn和Yn，0，2Xn和2，4An+1离子是同量异位的。 为了区分这两种糖对，用氰基硼氢化钠还原麦芽七糖。 在CAD中检测到广泛的碎片离子，Y离子的丰度最高。“热”ECD提供类似于CAD的裂解，但具有较少的交叉环裂解。 钠化和全甲基化N-连接支链聚糖：钠化和全甲基化高甘露糖和复合型（去唾液酸、二唾液酸双触角）N-连接聚糖的CAD和“热”ECD提供了补充结构信息。 用CAD中的B、Y离子和“热”ECD中的C、Z离子确认了这些聚糖的序列。 分支、组成和连锁信息通过交叉环裂解（CAD中的A型和“热”ECD中的互补A和X对）来确定。 内部碎片在CAD中特别常见，但也发生在ECD中。 需要更高的碰撞能量来将唾液酸化聚糖的主链片段化至与其脱唾液酸对应物相同的程度。 钠化和全甲基化二唾液酸化双触角N-连接聚糖的三重电荷分子离子丰富，并被“热”ECD片段化，产生大量片段离子（糖苷和互补交叉环裂解对），以充分确认其序列、分支和连接分配。 较大的高甘露糖型聚糖如GlcNAc 2 Man 7 -9通过ECD的片段化仍然是困难的，但是可以通过活化离子ECD来改善。 天然直链和支链聚糖的EDD：天然直链和支链N-连接聚糖的EDD实验比CAD产生更多的交叉环裂解;这些裂解可用于区分异构体。已出版了ECD手稿，EDD手稿已提交，目前正在收到初步审查后进行修订。