Zaia Joseph, Costello Catherine E
Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, R-806, Boston, Massachusetts 02118, USA.
Anal Chem. 2003 May 15;75(10):2445-55. doi: 10.1021/ac0263418.
The structural characterization of heparin-like glycosaminoglycans (HLGAGs) is a major challenge in glycobiology. These linear, sulfated oligosaccharides are expressed on animal cell surfaces, in extracellular matrixes, basement membranes, and mast cell granules and bind with varying degrees of specificity to families of proteases, growth factors, chemokines, and blood coagulation proteins. Cell surface HLGAGs bind growth factors and growth factor receptors and serve as coreceptors in these interactions. Understanding of the mechanism and regulation of growth factor-receptor binding requires efficient determination of cell surface HLGAG structures and the variations in their expression in response to the cellular environment. The solution to this problem entails rapid, sensitive structural analysis of these molecules. To date, HLGAG sequencing requires multistep processes that combine chemical and enzymatic degradation with gel-based or mass spectrometry-based detection systems. Although tandem mass spectrometry has revolutionized proteomics, the fragility of sulfate groups has limited its usefulness in the analysis of HLGAGs. This work demonstrates that tandem mass spectrometry can be effectively used to determine HLGAG structures while minimizing losses of SO3. First, collision-induced dissociation (CID) is shown to produce abundant backbone cleavage ions for HLGAG oligosaccharides, provided that most sulfate groups are deprotonated. Fragmentation of different precursor ion charge states produces complementary data on the structure of the HLGAG. Second, calcium ion complexation of HLGAGs stabilizes the sulfate groups, increases the relative abundances of backbone cleavage ions, and decreases the abundances of ions produced from SO3 losses.
类肝素糖胺聚糖（HLGAGs）的结构表征是糖生物学中的一项重大挑战。这些线性硫酸化寡糖在动物细胞表面、细胞外基质、基底膜和肥大细胞颗粒中表达，并以不同程度的特异性与蛋白酶、生长因子、趋化因子和血液凝固蛋白家族结合。细胞表面HLGAGs与生长因子和生长因子受体结合，并在这些相互作用中作为共受体。了解生长因子-受体结合的机制和调节需要高效测定细胞表面HLGAG结构及其在细胞环境响应中的表达变化。解决这个问题需要对这些分子进行快速、灵敏的结构分析。迄今为止，HLGAG测序需要多步过程，将化学和酶降解与基于凝胶或质谱的检测系统相结合。尽管串联质谱已经彻底改变了蛋白质组学，但硫酸根基团的脆弱性限制了其在HLGAG分析中的应用。这项工作表明，串联质谱可以有效地用于确定HLGAG结构，同时将SO3的损失降至最低。首先，碰撞诱导解离（CID）显示可为HLGAG寡糖产生丰富的主链裂解离子，前提是大多数硫酸根基团去质子化。不同前体离子电荷状态的碎片化产生了关于HLGAG结构的互补数据。其次，HLGAGs与钙离子络合可稳定硫酸根基团，增加主链裂解离子的相对丰度，并降低由SO3损失产生的离子丰度。
