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: 7955954
• 负责人: JOSEPH ZAIA
• 金额: $ 1.89万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955954
• 关键词: Acetylglucosamine; Amides; Area; Binding Proteins; Biology; Blood coagulation; Cattle; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Custom; Dalteparin; Databases; Effectiveness; Enoxaparin; Event; Family suidae; Funding; Glucuronic Acids; Glycosaminoglycans; Grant; Heparin; Heparin Lyase; Heparinoids; Heparitin Sulfate; Institution; Intestinal Mucosa; Ions; Kidney; Knowledge; Low-Molecular-Weight Heparin; Mass Spectrum Analysis; Medicine; Molecular Sieve Chromatography; Oligosaccharides; Pathogenesis; Peptides; Pharmaceutical Preparations; Phase; Polysaccharides; Reaction; Research; Research Personnel; Resources; Sampling; Source; Structure; System; United States National Institutes of Health; Work; aqueous; base; design; liquid chromatography mass spectrometry; meter; polymerization; polysulfated glycosaminoglycan

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. Porcine intestinal mucosa derived heparin was partially digested (30% reaction completion) with heparin lyase I. The resultant oligosaccharides were separated on a 1.75-meter size exclusion chromatography (SEC) column and pooled based on degree of polymerization. Bovine kidney HS was partially digested (10 or 20% reaction completion) to oligosaccharides with a mixture of heparin lyases I, II, and III. HS oligosaccharides as well as the low molecular weight heparin drugs Fragmin and Lovenox were purified by SEC-HPLC using a Superdex Peptide column. Samples were injected onto a custom-packed amide make-up flow HPLC-chip that was interfaced with an Agilent 6520 QTOF. Glycan compositions were assigned using a Microsoft Excel database and abundances calculated based on integrated peak area in the MS mode.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肝素和硫酸肝素(HS)是由交替的葡萄糖糖醛酸和N-乙酰氨基葡萄糖单元组成的糖胺多聚糖(GAG)。这些分子最广为人知的是它们能够结合蛋白质并调节细胞事件，如分化、凝血、发病和增殖。对这些事件的充分理解需要对GAG结构的了解。尽管芯片亲水作用色谱柱(HILIC)是一种强有力的在线LC/MS分离手段，但负离子喷雾在高含量水相流动相中变得不稳定。这项工作证明了一种芯片的有效性，该芯片采用柱后添加的补充液相流设计，能够在整个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著增加。 用肝素裂解酶I对猪肠黏膜肝素进行部分消化(反应完成率为30%)，得到的低聚糖在1.75m的尺寸排除色谱柱(SEC)上分离，并按聚合度合并。用肝素裂解酶I、II和III的混合物将牛肾HS部分消化(反应完成率为10%或20%)为寡糖。HS寡糖以及低分子肝素药物Fragmin和Lovenox用Superdex多肽柱通过SEC-HPLC纯化。样品被注入到定制包装的酰胺化妆流动高效液相芯片上，该芯片与Agilent 6520 QTOF接口。使用Microsoft Excel数据库指定多糖组成，并在MS模式下基于积分峰面积计算丰度。