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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 CAD，“热” ECD和EDD已被用来研究线性和分支聚糖的碎裂模式。通过在定制的Esi-ftiCr MS中提供的CAD和“热” ECD实验分析了二氧化基聚糖，并且所得的光谱提供了互补的结构信息。 CAD产生了主要的B和Y片段离子，而C和Z产品主导了“热” ECD产生了主要的C和Z离子。 在CAD产生的光谱中存在A型跨环裂解，而互补的A-和X型对是由“热” ECD引起的。 尤其是0,4AN和3,5AN离子定义了上部分支的连锁位置。 在CAD中观察到的内部碎片比在“热” ECD光谱中观察到更多。由于在负模式下，在负模式下形成更稳定的喷雾剂，因此对天然聚糖进行了EDD实验，以负模式进行。它产生的跨环裂解比CAD更多。天然聚糖，包括从糖蛋白中释放的聚糖，使用我们的标准方案进行了苄苄基化化。结果在此处简要汇总。二甲基化的线性麦芽醇 - 寡糖：同胞和苄苄苄化线性（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），跨环裂解（A和X离子）以及内部裂解（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离子是同型的。 为了区分对，通过氰基羟基氢化钠降低了厌食症。 在CAD中检测到广泛的片段离子，并且Y离子的丰度最高。 “热” ECD提供了类似于CAD的切割，尽管交叉环的切割较少。 在同胞和苄苄苄化的高甘露糖和复杂类型（ASIALO-，有害的Biantennary）N-连接的Glycans的N-连接的N-连接类型的N连接的N-Rin-Chlycans的N-连接的N-连接类型的N连接的N-连接的Glycans的N-链接ECD中，均二甲基化的N连接的分支聚糖：CAD和“热” ECD提供了互补的结构信息。 这些聚糖的序列通过“热” ECD中的CAD和C，Z离子的B，Y离子证实。 分支，组成和链接信息是通过跨环裂解（CAD中的A型和“热” ECD中的补充A和X对）确定的。 内部碎片在CAD中特别频繁，但在ECD中也发生。 需要较高的碰撞能量才能以与Asialo对应物相同的程度将硫化聚糖的骨架碎裂。 经过三倍的电荷分子离子的分子离子是二氧化和苄苄磷酸化的双胞tentenary n-链接聚糖的大量，并且被“热” ECD产生广泛的片段离子（糖苷和互补对）的碎片，以完全确认其序列，分支和链接分支和分支分支和分支分支和分支分支和分支分支。 ECD较大的高甘露糖型聚糖（例如GlcNAC2Man7-9）的碎片仍然很困难，但可以通过激活的离子ECD改善。 天然线性和分支聚糖的EDD：天然线性和分支N连接的聚糖上的EDD实验比CAD产生的交叉环裂解更多。这些切割可用于区分异构体。 ECD手稿发表了，EDD手稿已提交，并且在收到初步审查后目前正在修订。