DETERMINATION OF GLYCOSYLATION SITES OF GLYCOPROTEINS

我们正在继续制定明确的程序，以 鉴定那些糖肽的丝氨酸和苏氨酸残基 有固定的糖链。在这种方法中，糖肽是 用链霉酶消化，它以随机的方式裂解肽骨架 时尚。消化后的混合物用MALDI-MS分析 消化5、10和15分钟。这些光谱包含一系列 识别糖基化位点的多肽和糖多肽峰。 我们的链霉蛋白酶消化/MALDI-TOF程序的主要优点 用于识别糖基化位点的是它的速度(不到30分钟) 和敏感性(只需要1-10皮摩尔的糖肽)。 此外，该方法还适用于确定地块的位置 其他翻译后修饰，包括磷酸化， 硫酸盐化、二硫键和乙酰化。这个项目的目标是 填补目前分析方法的空白，能够 从亚纳摩尔量确定O-连接的糖基化位点 一种糖肽。

We are continuing to develop procedures for unambiguously identifying those serine and threonine residues of glycopeptides that have glycosyl chains attached. In this approach, the glycopeptide is digested with pronase, which cleaves the peptide backbone in a random fashion. The digestion mixtures are analyzed by MALDI-MS after digestion for 5, 10, and 15 min. These spectra contain a series of peptide and glycopeptide peaks that identify the glycosylation site. The primary advantages of our pronase digestion/MALDI-TOF procedure for identifying glycosylation sites are its speed (less than 30 min) and sensitivity (requiring only 1-10 picomoles of glycopeptide). Furthermore, this approach is suitable for determining the sites of other post-translational modifications, including phosphorylation, sulfation, disulfide bonds, and acetylation. This project is aimed at filling the current void of analytical procedures capable of determining O-linked glycosylation sites fro m sub-nanomole quantities of a glycopeptide.