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

使用补充流芯片改进类肝素的 HILIC LC/MS 分析

• 批准号: 8365549
• 负责人: JOSEPH ZAIA
• 金额: $ 2.62万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365549
• 关键词: Acetonitriles; Acetylglucosamine; Ammonium; Binding Proteins; Biology; Blood coagulation; Charge; Chromatography; Data; Dissociation; Effectiveness; Event; Family suidae; Funding; Glucuronic Acids; Glycosaminoglycans; Grant; Heparin; Heparin Lyase; Heparinoids; Heparitin Sulfate; High Pressure Liquid Chromatography; Incidence; Inorganic Sulfates; Intestines; Ions; Knowledge; Mass Spectrum Analysis; Medicine; Mucous Membrane; National Center for Research Resources; Oligosaccharides; Pathogenesis; Phase; Physiologic pulse; Polymers; Principal Investigator; Process; Protons; Publications; Research; Research Infrastructure; Resources; Running; Solutions; Source; Structure; System; Time; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Update; Vertebral column; Work; aqueous; base; cost; design; improved; ionization; liquid chromatography mass spectrometry; novel; polymerization; polysulfated glycosaminoglycan; protein complex; response

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 肝素和硫酸乙酰肝素 (HS) 是由交替的葡萄糖醛酸和 N-乙酰葡萄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）的能力而得到广泛认可。 充分理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) 是在线 LC/MS 分离 GAG 的强大方法，但负离子喷雾在高百分比水性流动相中变得不稳定。 这项工作证明了通过柱后添加补充 LC 流而设计的芯片的有效性，可在整个 HILIC 梯度中实现稳定的电喷雾。 其结果是系统的喷雾稳定性和可有效分析的分析物范围显着增加。 使用 CID 分析硫酸乙酰肝素的质谱方法受到 SO3 占主导地位的中性损失的限制，因此缺乏前体离子的糖苷和跨环裂解。由于前体离子中的移动质子促进了硫酸盐的损失，因此一种可能的解决方案是在 ESI 过程中增强电荷态，以减少可用的质子。环丁砜和其他试剂已被证明可以增加正模式下蛋白质复合物的电荷状态。 在本研究中，我们证明环丁砜能够提高负模式下 HS 寡糖的 ESI 效率和电荷状态。我们通过在在线 LC/MS 运行期间的指定时间段通过新型柱后流路芯片脉冲环丁砜来实现这一点。 猪肠粘膜硫酸乙酰肝素被肝素裂解酶I、II和III一起部分消化，得到一定聚合物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芯片，通过在线亲水相互作用色谱 (HILIC)-LC/MS（带芯片立方体接口的安捷伦 QTOF）分析 dp4-6 寡糖。乙腈中的 105 mM 环丁砜在大多数 dp4-6 的洗脱时间通过定制安捷伦 HPLC 芯片上的尾吹流脉冲 1 分钟，该芯片具有额外的 100 nl 储液环，用于在分析柱之后和喷雾之前进行添加剂脉冲。 环丁砜脉冲使硫酸肝素寡糖的电离响应增加了 5 至 10 倍。对于硫酸化程度最高的寡糖，这种效果似乎最为明显。 添加 105 mM 环丁砜可使高电荷态下高度硫酸化寡糖的强度增加约 20 倍。主要电荷态从 2- 转变为 4-。还观察到 dp6 寡糖的最大电荷态 5-，这是使用不添加环丁砜的 HILIC LC/MS 时未观察到的电荷态。除了电荷态增加之外，含有铵加合的离子的丰度也显着降低。 高度硫酸化寡糖电荷态的增加还有助于将它们在m/z上与电荷态未增加的不太高度硫酸化的寡糖分开。 结果，减少了重叠电荷状态的发生率。 这些数据显示了对硫酸乙酰肝素寡糖进行更有效的串联质谱分析的潜力，因为较高的电荷态可能会产生更丰富的主链和跨环裂解产物离子。 进度更新： 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，我们期望使用 MUF 芯片添加环丁砜以促进 HS 寡糖串联 MS。 这与 HS 寡糖衍生化相结合，可能会最大限度地利用此类化合物的碰撞诱导解离产生的信息。