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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 利用CAD、“热”ECD和EDD研究了线状和支链糖链的碎裂模式。在定制的ESI-FTICR MS中，通过CAD和“热”ECD实验分析了碱化和全甲基化的葡聚糖，得到的光谱提供了补充的结构信息。CAD产生了主要的B和Y碎片离子，而C和Z产物占主导地位，而热的ECD产生了主要的C和Z离子。在CAD产生的光谱中存在A型交叉环解理，而热ECD产生互补的A型和X型对。尤其是0，4An和3，5An离子确定了上支链的连接位置。在CAD中观察到比在“热”ECD谱中更丰富的内部碎片。由于酸性多糖在负模式下形成的喷雾比在正模式下更稳定，所以对天然多糖的EDD实验是在负模式下进行的，它产生了比CAD更多的交叉环切割。使用我们的标准方案对天然多糖，包括那些从糖蛋白中释放出来的多糖进行了全甲基化。本文对研究结果进行了简要总结。碱化和全甲基线性麦芽低聚糖：碱化和全甲基线性(GLC)6-(GLC)9的碎裂模式非常相似。观察到糖苷裂解(B、Y、C和Z离子)、跨环裂解(A和X离子)和内部裂解(B/Y和C/Y离子)。由于其高度对称的结构，Bn和Zn，CN和Yn，0，2Xn和2，4An+1离子是等压的。为了区分这些对，用氰基硼氢化钠还原了麦芽七糖。在CAD中检测到大量碎片离子，其中Y离子丰度最高。“热”ECD提供类似于CAD的卵裂，但具有较少的交叉环裂。碱化和全甲基化N-连接的支链多糖：碱化和全甲基化的高甘露糖和复合型(去唾液酸双天线)N-连接的糖链上的CAD和“热”ECD提供了补充的结构信息。CAD中的B、Y离子和热的ECD中的C、Z离子证实了这些糖链的序列。分支、组成和连接信息由交叉环切割(CAD中的A型，热的ECD中的互补A和X对)决定。内部碎片在CAD中尤其常见，但在ECD中也会出现。需要更高的碰撞能量才能将唾液酸化的葡聚糖的主链碎裂到与其asialo对应物相同的程度。碱化和全甲基化的二唾液酸联二天线N-连接的多糖的三电荷分子离子丰富，并被产生大量碎片离子(糖苷和互补的交叉环裂解对)的“热”ECD裂解，以充分确认其序列、分支和连接指定。较大的高甘露糖型葡聚糖如GlcNAc2Man7-9的ECD裂解仍然很困难，但活化离子ECD可以改善。天然直链和支链多糖的EDD：对天然直链和支链N-连接的葡聚糖的EDD实验产生了比CAD更多的十字环裂解；这些裂解可用于区分异构体。ECD手稿已经出版，EDD手稿已经提交，目前正在收到初步审查后进行修订。