Comprehensive chromatographic profiling and structural analysis of key anticoagulant components in enoxaparin

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
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Abstract

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding.
对依诺肝素中的主要抗凝成分进行全面色谱分析和结构分析。
肝素是临床上使用最广泛的抗凝剂,其中依诺肝素是最重要的低分子量肝素(LMWHs)之一。本研究使用了抗凝血酶 III(ATIII)亲和柱。用制备型尺寸排阻色谱法(SEC)制备的不同尺寸的依诺肝素及其寡糖通过 ATIII 亲和柱进行分馏。使用强阴离子交换(SAX)色谱法对每种寡糖的不同亲和性馏分进行了分析。在进行质谱(MS)分析之前,每个峰都会自动转移到一个 SEC 柱上进行脱盐,从而利用多心切片(MHC)二维 LC-MS 系统(SAX-SEC-MS)进行结构鉴定。利用 MHC 二维液相色谱系统(SEC-SAX)进一步分析了依诺肝素的高亲和力馏分。通过大小分馏和亲和性分馏制备的高亲和性寡糖的 SAX 图谱与直接 SEC-SAX 分析获得的结果一致。通过人工收集寡糖后进行双糖分析,进一步阐明了烯诺肝素中几种高亲和性六糖的可能序列以及高亲和性八糖和十糖的结构域组成。这项工作加深了人们对依诺肝素结构特征的了解,为提高依诺肝素的质量以及确定肝素/LMWHs 中有助于蛋白质结合的关键结构基团提供了一种潜在的方法。
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来源期刊
Journal of Chromatography A
Journal of Chromatography A 化学-分析化学
CiteScore
7.90
自引率
14.60%
发文量
742
审稿时长
45 days
期刊介绍: The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.
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