FRAGMENTATION STUDIES OF SIALYLATED OLIGOSACCHARIDES

唾液酸化低聚糖的断裂研究

• 批准号: 7369307
• 负责人: JOSEPH ZAIA
• 金额: $ 3.2万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369307
• 关键词: FRAGMENTATION; STUDIES; SIALYLATED; OLIGOSACCHARIDES

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. Introduction: Sialylation of oligosaccharides affects the fragmentation and the energetics of dissociation. Using dissociation energy profiles of asialo- and sialylated oligosaccharides, the mechanism of glycosidic bond cleavage was observed to depend upon the number of sialic acid residues. Analysis by negative ion nanospray-MS/MS on a quadrupole-orthogonal time-of-flight instrument revealed that sialylated ions require substantially more energy to undergo fragmentation than asialo-forms. In asialo forms, deprotonation of a hydroxyl oxygen destabilized the glycosidic bond, but with sialylated forms, the charge resided on the sialic acid, and more energy was required to fragment the glycosidic bond. The energetic nature of deprotonated asialo-glycan ions facilitates glycosidic bond fragmentation during CID, while deprotonated sialylated glycan ions are lower in energy and resist glycosidic fragmentation. Methods: Lewis A, Lewis X, Sialyl Lewis A, Sialyl Lewis X, LNT, LNnT, DSLNT, LST-a, LST-d, and LST-c were analyzed using negative ion nanospray on an ABI Pulsar i QStar QoTOF MS. Solutions were sprayed from 30% MeOH in water with 0.1% ammonium hydroxide at concentrations between 1 pM and 10 pM. Tandem mass spectra of either singly or doubly charged precursor ions were acquired at varying collision energies, -2.5V to -47.5V. Spectra were collected for one minute and averaged for analysis. Breakdown curves for ions of interest were plotted as the percentage of the total ion intensity versus the collision energy. The threshold for peak selection was held to 5% of the intensity of the base peak. Results: Sialylation of milk oligosaccharides significantly changes the dissociation energetics of this class of compounds. In negative ion nanospray, the energy required to deplete the precursor intensity by 50% of neutral LNT and LNnT is roughly -12V; however the sialylated forms, LST-a and LST-d respectively, are much more stable and require roughly -37V to fragment to the same extent. LNT and LNnT produce abundant Y2 and Y3 ions at low energies, indicating the destabilization of the glycosidic bond, while sialylated structures produce abundant B1 ions showing the loss of a sialic acid residue as well as higher energy C ions. When comparing the energetics of asialo- and sialylated forms of Lewis antigens, large differences were also seen in the collision energy required to fragment the precursor ion to 50% of its initial intensity. Lewis X and Lewis A required -6V, while the sialylated forms required four times the amount of energy, -27-29V. Charge is produced in neutral sugars by deprotonation of a hydroxyl oxygen during the ionization process. This anion is evidently energetic enough to undergo glycosidic bond cleavage at relatively low collision energies. As the ions have gained energy during the ionization process, the energy for complete fragmentation during CID is minimal. The sialylated sugars have the negative charge residing on the terminal sialic acid, remote from the glycosidic oxygen; therefore, to cause glycosidic bond fragmentation energy needs to be added to the system in order to induce homolytic bond cleavage or deprotonate one of the hydroxyl oxygens. Sialylated sugars thus require more energy during the CID process to induce glycosidic cleavages.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。简介：寡糖的唾液酸化影响断裂和解离能量。使用脱唾液酸寡糖和唾液酸化寡糖的解离能谱，观察到糖苷键断裂的机制取决于唾液酸残基的数量。在四极杆正交飞行时间仪器上进行的负离子纳喷雾 MS/MS 分析表明，唾液酸化离子比脱唾液酸离子需要更多的能量才能发生碎裂。在无唾液酸形式中，羟基氧的去质子化使糖苷键不稳定，但在唾液酸化形式中，电荷驻留在唾液酸上，并且需要更多的能量来断裂糖苷键。去质子化的脱唾液酸聚糖离子的能量性质有利于 CID 期间糖苷键的断裂，而去质子化的唾液酸聚糖离子的能量较低并能抵抗糖苷断裂。方法：使用负离子纳喷雾在 ABI Pulsar i QStar QoTOF MS 上分析 Lewis A、Lewis X、Sialyl Lewis A、Sialyl Lewis X、LNT、LNnT、DSLNT、LST-a、LST-d 和 LST-c。将溶液从 30% MeOH 水溶液与 0.1% 氢氧化铵中喷洒，浓度在 1 pM 至 10 pM 之间。在不同的碰撞能量（-2.5V 至 -47.5V）下获得单电荷或双电荷前体离子的串联质谱。收集光谱一分钟并取平均值以供分析。将感兴趣的离子的击穿曲线绘制为总离子强度与碰撞能量的百分比。峰选择的阈值保持为基峰强度的 5%。 结果：乳低聚糖的唾液酸化显着改变了此类化合物的解离能量。在负离子纳米喷雾中，将中性LNT和LNnT的前体强度消耗50%所需的能量大致为-12V；然而，唾液酸化形式 LST-a 和 LST-d 分别更加稳定，需要大约 -37V 才能裂解到相同程度。 LNT 和 LNnT 在低能量下产生丰富的 Y2 和 Y3 离子，表明糖苷键不稳定，而唾液酸化结构产生丰富的 B1 离子，表明唾液酸残基以及更高能量的 C 离子的损失。当比较Lewis抗原的脱唾液酸形式和唾液酸化形式的能量学时，将前体离子碎裂至其初始强度的50%所需的碰撞能量也存在很大差异。 Lewis X和Lewis A需要-6V，而唾液酸化形式需要四倍的能量，-27-29V。中性糖在电离过程中通过羟基氧的去质子化产生电荷。该阴离子显然具有足够的能量，能够在相对较低的碰撞能量下发生糖苷键断裂。由于离子在电离过程中获得了能量，因此 CID 期间完全碎裂的能量很小。唾液酸化糖的负电荷位于末端唾液酸上，远离糖苷氧；因此，为了引起糖苷键断裂，需要向系统中添加能量，以诱导均裂键断裂或使羟基氧之一去质子化。因此，唾液酸化糖在 CID 过程中需要更多能量来诱导糖苷裂解。