Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling
Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling
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DOI：
10.1074/jbc.m804288200
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发表时间：
2008-11-28
影响因子：
4.8
通讯作者：
Esko, Jeffrey D.
中科院分区：
文献类型：
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作者：
Lawrence, Roger;Olson, Sara K.;Esko, Jeffrey D.
为了便于糖胺聚糖的定性和定量分析，我们用含有苯胺的稳定同位素(C-12(6)和C-13(6))标记了裂解酶生成的二糖的还原末端。由于不同的同位素标记对色谱保留时间没有影响，但可以由质量检测器区分，因此不同的同位素标记的样品可以同时通过液相色谱/质谱仪进行比较，并通过与已知标准量的混合物进行定量。该技术适用于所有类型的糖胺多聚糖，其灵敏度仅受可用质谱计类型的限制。我们使用商业肝素和硫酸角蛋白以及从突变和野生型中国仓鼠卵巢细胞中分离的硫酸肝素验证了该方法，并选择了突变和野生型小鼠的组织。与现有技术相比，这种新方法提供了更可靠、更灵敏的糖胺聚糖双糖组成的定量评估手段，使我们能够比较普通九头蛇、果蝇、秀丽线虫和哺乳动物细胞的软骨素和硫酸乙酰肝素的组成。我们的结果表明，这些生物之间的糖胺多聚糖结构存在显著差异，这可能代表了进化上不同的功能基序。
To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (C-12(6) and C-13(6)). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.