Drummond K J, Yates E A, Turnbull J E
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Proteomics. 2001 Feb;1(2):304-10. doi: 10.1002/1615-9861(200102)1:2<304::AID-PROT304>3.0.CO;2-B.
The sequencing of heparan sulfate oligosaccharides has recently become possible using integral Glycan Sequencing, which utilizes a combination of chemical and enzymatic degradation steps followed by polyacrylamide gel electrophoresis. This technique has previously employed the fluorescent label, anthranilic acid, and has been used to sequence low nmol amounts of purified saccharides. Here, we present an improved method, which uses the alternative label, 7-aminonapthalene-1,3-disulfonic acid, the reducing agent sodium triacetoxyborohydride and optimizes the nitrous acid step in heparin/heparan sulfate degradation. These improvements increase the sensitivity at least ten-fold taking the amount of starting material required into the pmol range. We show that this label is compatible with the integral glycan sequencing methodology and demonstrate its application to the sequencing of chemically modified heparin derivatives. Advances in sequencing techniques for heparan sulfate saccharides will permit detailed structure-function studies and will in the future underpin novel proteomics-based approaches aimed at studying their diverse functional roles as protein regulators.
硫酸乙酰肝素寡糖的测序最近通过完整聚糖测序得以实现，该方法利用化学和酶促降解步骤的组合，随后进行聚丙烯酰胺凝胶电泳。此技术先前使用荧光标记邻氨基苯甲酸，并已用于对低纳摩尔量的纯化糖类进行测序。在此，我们提出一种改进方法，该方法使用替代标记7-氨基萘-1,3-二磺酸、还原剂三乙酰氧基硼氢化钠，并优化肝素/硫酸乙酰肝素降解中的亚硝酸步骤。这些改进将灵敏度提高了至少十倍，使所需起始材料的量进入皮摩尔范围。我们表明该标记与完整聚糖测序方法兼容，并证明了其在化学修饰的肝素衍生物测序中的应用。硫酸乙酰肝素糖类测序技术的进步将允许进行详细的结构-功能研究，并将在未来为旨在研究其作为蛋白质调节剂的多种功能作用的新型蛋白质组学方法提供支持。
