DETERMINATION OF GLYCOSYLATION SITES OF GLYCOPROTEINS

我们正在继续制定程序， 鉴定那些糖肽的丝氨酸和苏氨酸残基， 连接有糖基链。 在这种方法中，糖肽是 用链霉蛋白酶消化，链霉蛋白酶以随机的方式切割肽骨架， 时尚. 消化混合物通过MALDI-MS分析， 消化5、10和15分钟。这些光谱包含一系列 识别糖基化位点的肽和糖肽峰。 我们的链霉蛋白酶消化/MALDI-TOF程序的主要优点 鉴定糖基化位点的最佳条件是其速度（小于30 min） 和灵敏度（仅需要1-10皮摩尔的糖肽）。 此外，该方法适用于确定 其它翻译后修饰，包括磷酸化， 硫酸化、二硫键和乙酰化。 该项目旨在 填补了目前分析方法的空白， 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.