IMPROVED HILIC LC/MS ANALYSIS OF HEPARINOIDS USING A MAKEUP FLOW CHIP

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 肝素和硫酸乙酰肝素（HS）是由交替的葡萄糖醛酸和N-乙酰葡糖胺单元组成的糖胺聚糖（GAG）。 这些分子因其结合蛋白质和调节细胞事件如分化、凝血、发病机制和增殖的能力而被最广泛地认可。 充分了解这些事件需要了解GAG结构。 尽管基于芯片的亲水相互作用色谱（HILIC）是用于在线LC/MS分离GAG的有力手段，但负离子喷雾在高百分比水移动的相中变得不稳定。 这项工作证明了设计为柱后添加补充LC流的芯片的有效性，以在整个HILIC梯度中实现稳定的电喷雾。 结果是系统具有显著增加的喷雾稳定性和可以有效分析的分析物范围。 使用CID分析硫酸乙酰肝素的质谱方法受到SO 3的主要中性损失的限制，因此缺乏前体离子的糖苷和交叉环裂解。由于前体离子中的移动的质子促进了硫酸根的损失，因此一种可能的解决方案是在ESI过程期间增强电荷状态以减少可用的质子。环丁砜和其他药剂已被证明在正模式下增加蛋白质复合物的电荷状态。 在这项研究中，我们证明了环丁砜是能够增加的ESI效率和HS寡糖在负模式下的电荷状态。我们通过在线LC/MS运行期间在指定时间段经由新型柱后流动芯片脉冲环丁砜来实现这一点。 用肝素裂解酶Ⅰ、Ⅱ和Ⅲ部分消化猪肠粘膜硫酸乙酰肝素，得到具有一定聚合范围的随机组成的寡糖。通过将消化产物施加到SEC柱来收集聚合度（dp）4-6级分。 使用定制的脉冲补充流动芯片通过在线亲水相互作用色谱（HILIC）-LC/MS（具有Chip cube接口的Agilent QTOF）分析dp 4 -6寡糖。在大多数dp 4 -6的洗脱时间下，将乙腈中的105 mM环丁砜通过定制的Agilent HPLC芯片上的补充流脉冲1分钟，所述定制的Agilent HPLC芯片具有额外的100 nl储存器回路，用于在分析柱之后和喷雾之前进行添加剂脉冲。 脉冲环丁砜增加了硫酸肝素寡糖的电离响应的一个因素，范围从5到10。 对于硫酸化程度最高的低聚糖，这种影响似乎最为明显。 添加105 mM环丁砜使高电荷状态下高度硫酸化寡糖的强度增加约20倍。主要电荷态由2-转变为4-。还观察到dp 6寡糖的最大电荷状态5-，当使用不添加环丁砜的HILIC LC/MS时未观察到的电荷状态。除了电荷状态的增加，含有铵加合物的离子的丰度显着降低。 高度硫酸化的寡糖的电荷状态的增加也用于将它们在m/z中与电荷状态未增加的较不高度硫酸化的寡糖分离。 结果，减少了重叠电荷状态的发生。 数据显示，由于较高的电荷状态可能产生更丰富的骨架和跨环裂解产物离子，因此有可能实现乙酰肝素硫酸化低聚糖的更有效串联MS。 最新进展： 我们正在整理一份关于使用脉冲MUF芯片改进GAG寡糖串联质谱的出版物。 展望未来，我们希望使用MUF芯片添加环丁砜以促进HS寡糖串联MS。这与HS寡糖衍生化结合，可能会最大限度地提高使用碰撞诱导解离对该化合物类别产生的信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) comprised of alternating glucuronic acid and N-acetylglucosamine units. These molecules are most widely recognized for their ability to bind proteins and modulate cellular events such as differentiation, blood coagulation, pathogenesis, and proliferation. Full understanding of these events requires knowledge of GAG structure. Although chip-based hydrophilic interaction chromatography (HILIC) is a powerful means of separating GAGs for on-line LC/MS, the negative-ion spray becomes unstable in high percent aqueous mobile phases. This work demonstrates the effectiveness of a chip designed with post-column addition of makeup LC flow to enable stable electrospray throughout the HILIC gradient. The result is a system with substantially increased spray stability and range of analytes that may be effectively analyzed. The mass spectrometric approach of analyzing heparan sulfate using CID has been limited by the dominant neutral loss of SO3 and consequently, the lack of glycosidic and cross-ring cleavage of the precursor ions. Since the loss of the sulfate is facilitated by mobile protons in the precursor ions, one possible solution would be to enhance the charge states during the ESI process to reduce available protons. Sulfolane and other agents have been shown to increase the charge states of protein complexes in the positive mode. In this study we demonstrate that sulfolane is able to increase both the ESI efficiency and charge states of HS oligosaccharide in the negative mode. We accomplish this by pulsing sulfolane via a novel post-column flow chip at designated time period during an online LC/MS run. Porcine intestine mucosa heparan sulfate was partially digested by heparin lyase I, II and III together to give random composition of oligosaccharides of a certain polymer range. The degree of polymerization (dp) 4-6 fraction was collected by applying the digest products to SEC column. The dp4-6 oligosaccharides were analyzed by online hydrophilic interaction chromatography (HILIC)-LC/MS (Agilent QTOF with Chip cube interface) using a customized pulsed makeup flow chip. 105 mM sulfolane in acetonitrile was pulsed for 1 min at the elution time of most dp4-6 through a makeup flow on a customized Agilent HPLC-chip that has an extra 100 nl reservoir loop for additive pulsing after the analytical column and before spraying. Pulsing of sulfolane increased the ionization response for heparin sulfate oligosaccharides by a factor ranging from 5 to 10. The effect appears to be most pronounced for the most highly sulfated oligosaccharides. 105 mM sulfolane addition increased the intensity of highly sulfated oligosaccharides at higher charge state by approximately 20-fold. The dominant charge state was shifted from 2- to 4-. A maximum charge state 5- for dp6 oligosaccharides was also observed, a charge state that has not been observed when using HILIC LC/MS without sulfolane addition. In addition to the increase in charge state, ions containing ammonium adduction were significantly lower in abundance. The increase in the charge state of highly sulfated oligosaccharides also served to separate them in m/z from less highly sulfated oligosaccharides whose charge state were not increased. As a result, the incidence of overlapping charge states is reduced. The data show potential to enable more effective tandem MS of heparan sulfated oligosaccharides because higher charge states are likely to produce more abundant backbone and cross ring cleavage product ions. Progress update: We are putting together a publication on the use of the pulsed MUF chip for improved tandem MS of GAG oligosaccharides. Going forward, we expect to use the MUF chip to add sulfolane to facilitate HS oligosaccharide tandem MS. This, in combination with HS oligosaccharide derivatization, is likely to maximize the information produced using collisional induced dissociation on this compound class.