MOBILE PROTONS IN NEGATIVE CID TANDEM MS OF SULFATED OLIGOSACCHARIDES

硫酸低聚糖负 CID 串联质谱中的移动质子

• 批准号: 7369297
• 负责人: JOSEPH ZAIA
• 金额: $ 2.03万
• 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/7369297
• 关键词: MOBILE; PROTONS; NEGATIVE; CID; TANDEM

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: Most classes of animal glycans are acidic, and the use of negative ionization is a natural choice. The aim of this work is to explain product ion fragmentation patterns for negatively charged acidic glycans. Such an understanding is important to develop tandem MS strategies in glycomics. This work shows how the presence of uronic acid residues influences the product ion patterns of sulfated glycosaminoglycan oligosaccharides. Uronic acid protons becomes mobile during the CID process and destabilize glycosidic bonds. In the absence of such mobile protons, the ions are significantly more stable and require more energy for glycosidic cleavage. Both charge location and potentially mobile protons strongly influence product ion pattern for acidic oligosaccharides. Methods: Oligosaccharides were produced by digesting chondroitin sulfate with chondroitinase ABC or testicular hyaluronidase and purified by size exclusion chromatography using a Superdex Peptide column (Amersham Pharmacia). Oligosaccharides were methyl esterified using methanolic HCl. All mass spectra were acquired in nano-electrospray mode using a Applied Biosystems/Sciex Qstar Pulsar-i mass spectrometer. Samples were dissolved at a concentration of ~1 micromolar in 30% methanol and sprayed through uncoated borosilicate glass tips pulled to a 1 micrometer diameter orifice. Steady signals were typically observed with -1100 V spray potentials. Results: The presence of acidic groups (sulfate, phosphate, sialic acid) strongly influences the pattern of product ions resulting from CID of negative oligosaccharide ions. Tandem MS of chondroitin sulfate oligosaccharides of the form (?HexA)(HexA)n-1(GalNAcSulfate)n, where ?HexA = 4,5-unsaturated HexA, were acquired before and after methyl esterification of uronic acid residues. The native structures fragment under significantly less energetic conditions than do the methyl esterified glycans. Abundances of cross-ring cleavage ions increase for the methyl esterified relative to the native glycans. Chondroitin sulfate oligosaccharides lacking a ?-unsaturated uronic acid residue do not have cross-ring cleavage as a fragmentation channel. In their methyl esterified forms, these ions require approximately -22 V collision energy to reduce the precursor ion intensity by 50%. For the native structures, only -15 V collision energy is required. These results are consistent with the conclusion that, despite the deprotonated precursor ion, remaining carboxyl protons become delocalized as the ion temperature rises during the CID process and associate with glycosidic oxygen atoms. This association predisposes the glycosidic bond to scission, in a manner similar to that described for positively charged glycans. Methyl esterified precursor ions, lacking carboxylic protons, require significantly higher fragmentation energies than do native ions. The abundances of cross-ring cleavages increase because of the lack of mobile protons to destabilize glycosidic bonds. Both the location of negative charge and the presence of protons that become mobilized during the CID process strongly influence the observed product ion patterns. In the absence of mobile protons, acidic glycans resist glycosidic bond cleavage because charge is located distant from the glycosidic bonds. These results have clear implications regarding the design of on-line LC MS/MS conditions for glycomics experiments.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。导言：大多数种类的动物多糖都是酸性的，使用负电离是自然而然的选择。这项工作的目的是解释带负电荷的酸性多糖的产物离子碎裂模式。这样的理解对于开发糖组学中的串联MS策略是重要的。这项工作展示了糖醛酸残基的存在如何影响硫酸氨基寡糖的产物离子模式。糖醛酸质子在CID过程中变得可移动，并破坏糖苷键的稳定。在没有这种可移动质子的情况下，离子明显更稳定，需要更多的能量来进行糖苷切割。电荷位置和潜在的可移动质子都强烈影响酸性低聚糖的产物离子模式。方法：用硫酸软骨素酶ABC或睾丸透明质酸酶消化硫酸软骨素制备低聚糖，用Superdex多肽柱(Amersham Pharmacia)进行体积排阻层析纯化。低聚糖用甲醇盐酸甲酯化。使用应用生物系统/Sciex QStar Pulsar-I质谱仪以纳米电喷雾模式获得所有的质谱图。样品以~1微摩尔的浓度溶解在30%的甲醇中，通过未涂覆的硼硅酸盐玻璃喷嘴喷射到直径1微米的孔。在-1100V的喷雾电位下，通常可以观察到稳定的信号。结果：酸性基团(硫酸盐、磷酸盐、唾液酸)的存在强烈影响负寡糖离子的CID产物离子的模式。在糖醛酸残基甲酯化前后，得到了形式为(6)(6)(6)n-1(GalNAcSulate)n的硫酸软骨素低聚糖的串联MS，其中6=4，5-不饱和6。天然结构在能量显著低于甲酯化的糖聚糖的条件下碎裂。与天然多糖相比，甲基酯化的葡聚糖跨环裂解离子的丰度增加。缺乏β-不饱和糖醛酸残基的硫酸软骨素低聚糖不具有作为碎裂通道的交叉环裂解。在它们的甲酯化形式中，这些离子需要大约-22V的碰撞能量才能将前体离子强度降低50%。对于本征结构，只需要-15V的碰撞能量。这些结果与以下结论是一致的，即尽管存在去质子化的前体离子，但在CID过程中，随着离子温度的升高，剩余的羧基质子会离域，并与糖苷氧原子伴生。这种结合使糖苷键容易断裂，其方式类似于所描述的带正电的多糖。甲酯化的前体离子缺乏羧基质子，需要比天然离子高得多的碎裂能。由于缺乏破坏糖苷键稳定的可移动质子，跨环裂解的丰度增加。负电荷的位置和在CID过程中被动员的质子的存在都强烈地影响着观察到的产物离子模式。在没有可移动质子的情况下，酸性多糖抵抗糖苷键的断裂，因为电荷位于远离糖苷键的位置。这些结果对糖组学实验的在线LC-MS/MS条件的设计具有明显的指导意义。