FRAGMENTATION STUDIES OF SIALYLATED OLIGOSACCHARIDES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。简介： 低聚糖的唾液酸化作用影响其裂解和解离能。使用去唾液酸和唾液酸化寡糖的解离能谱，观察到糖苷键裂解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通过负离子纳米喷雾-MS/MS进行的分析显示，唾液酸化离子比去唾液酸形式需要更多的能量来进行碎片化。在去唾液酸形式中，羟基氧的去质子化使糖苷键不稳定，但对于唾液酸化形式，电荷驻留在唾液酸上，并且需要更多的能量来使糖苷键断裂。去质子化的无唾液酸聚糖离子的能量性质在CID期间促进糖苷键断裂，而去质子化的唾液酸化聚糖离子的能量较低并且抵抗糖苷断裂。研究方法： 在ABI Pulsar i QStar QoTOF MS上使用负离子纳米喷雾分析刘易斯A、刘易斯X、Sialyl刘易斯A、Sialyl刘易斯X、LNT、LNnT、DSLNT、LST-a、LST-d和LST-c。将溶液从含有0.1%氢氧化铵的30%MeOH水溶液以1 pM至10 pM的浓度喷雾。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用于分析。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。峰选择的阈值保持在基峰强度的5%。 结果如下： 牛奶低聚糖的唾液酸化显著改变了这类化合物的解离能。在负离子纳米喷雾中，将中性LNT和LNnT的前体强度消耗50%所需的能量约为-12V;然而，唾液酸化形式LST-a和LST-d分别要稳定得多，并且需要约-37V才能破碎到相同程度。LNT和LNnT在低能量下产生丰富的Y2和Y3离子，表明糖苷键的不稳定，而唾液酸化结构产生丰富的B1离子，表明唾液酸残基的损失以及较高能量的C离子。当比较刘易斯抗原的去唾液酸形式和唾液酸化形式的能量时，在将前体离子碎片化至其初始强度的50%所需的碰撞能量中也观察到很大的差异。刘易斯X和刘易斯A需要-6伏，而唾液酸化形式需要四倍的能量，-27-29伏。中性糖在电离过程中通过羟基氧的去质子化产生电荷。这种阴离子显然具有足够的能量，可以在相对较低的碰撞能量下进行糖苷键断裂。由于离子在电离过程中获得了能量，因此CID过程中完全碎裂的能量最小。唾液酸化糖具有位于远离糖苷氧的末端唾液酸上的负电荷;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裂解或使羟基氧之一去质子化。因此，唾液酸化糖在CID过程中需要更多的能量来诱导糖苷裂解。

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.