Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling
Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling
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DOI：
10.1093/glycob/cwl046
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发表时间：
2007-01-01
期刊：
影响因子：
4.3
通讯作者：
Zaia, Joseph
中科院分区：
文献类型：
--
作者：
Hitchcock, Alicia M.;Yates, Karen E.;Zaia, Joseph
关节软骨是一种高度特化的光滑结缔组织，其正常功能取决于细胞外基质的维持，所述细胞外基质由胶原、糖蛋白、蛋白聚糖（PG）和糖胺聚糖的整合组装组成。硫酸化和糖醛酸差向异构化的位置和程度不同的异构硫酸软骨素糖型在发育和疾病发作期间发挥特定和独特的功能作用。本工作介绍了一种新的糖胺聚糖提取方法，用于完整软骨组织中硫酸软骨素寡糖混合物的定量质谱分析。使用乙醇沉淀和酶促释放的组合，然后通过反相和强阴离子交换固相提取步骤，从完整的软骨样品中提取糖胺聚糖。提取的硫酸软骨素糖胺聚糖使用软骨素酶部分解聚，用2-邻氨基苯甲酸-d（4）（2-AA）标记，并在负离子模式下进行尺寸排阻色谱和在线电喷雾电离质谱检测。本文提出的方法能够同时测定幼年牛和成年人软骨样品中的硫酸盐位置和糖醛酸差向异构化。该方法应用于一系列的13个成人软骨外植体。测量的平均值的标准偏差平均为1.6。对于40%或更大的所有组合物，变异系数约为4%。这些结果表明，新方法具有足够的准确度，可以确定结缔组织中糖型的拓扑分布。
Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.