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: 8170920
• 负责人: JOSEPH ZAIA
• 金额: $ 1.85万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170920
• 关键词: Acetylglucosamine; Binding Proteins; Blood coagulation; Charge; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Effectiveness; Event; Funding; Glucuronic Acids; Glycosaminoglycans; Grant; Heparin; Heparinoids; Heparitin Sulfate; Inorganic Sulfates; Institution; Ions; Knowledge; Oligosaccharides; Pathogenesis; Phase; Physiologic pulse; Process; Protons; Research; Research Personnel; Resources; Solutions; Source; Structure; System; Time; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Work; aqueous; base; design; liquid chromatography mass spectrometry; novel; polysulfated glycosaminoglycan; protein complex

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. 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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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有柱后添加补充LC流的芯片的有效性，以实现整个HILIC梯度的稳定电喷雾。 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期间指定时间段通过新型柱后流动芯片脉冲环丁砜来实现这一目标