Use of a lectin affinity selector in the search for unusual glycosylation in proteomics
Use of a lectin affinity selector in the search for unusual glycosylation in proteomics
复制标题
DOI：
10.1016/s1570-0232(02)00671-2
复制
发表时间：
2002-12-25
影响因子：
3
通讯作者：
Regnier, FE
中科院分区：
文献类型：
--
作者：
Xiong, L;Regnier, FE
本文所述的工作目的是开发一种新的方法来鉴定具有特定糖基化类型的糖蛋白。本文证明了糖蛋白的n -糖基化位点可以通过(1)胰蛋白酶的蛋白水解，(2)凝集素亲和选择，(3)肽- n -糖苷酶F （PNGase F）在含有95% (H2O)-O-18的缓冲液中酶解去糖基化，产生2或4个氨基酸间隔的去糖基化肽对，(4)肽混合物的反相分离和MALDI质谱分析，(5)离子对的MS-MS测序。(6)通过数据库检索鉴定亲本蛋白。对这一过程进行了乳铁蛋白和乳蛋白中糖肽的选择和胎蛋白糖肽离子对的鉴定。通过PNGase水解(H2O)-O-18确定糖基化位点。在水解共轭物的过程中，Asn在O-18的掺入下转化为天冬氨酸残基。然而，观察到PNGase F将两个O-18结合到Asp残基的对羧基上。这表明水解至少是部分可逆的。(C) 2002 Elsevier Science B.V.版权所有
The purpose of the work described in this paper was to develop a new approach to the identification of glycoprotein with particular types of glycosylation. The paper demonstrates N-glycosylation sites in a glycoproteins can be identified by (1) proteolysis with trypsin, (2) lectin affinity selection, (3) enzymatic deglycosylation with peptide-N-glycosidase F (PNGase F) in buffer containing 95% (H2O)-O-18, which generates deglycosylated peptide pairs separated by 2 or 4 amu, (4) reversed-phase separation of the peptide mixture and MALDI mass analysis, (5) MS-MS sequencing of the ion pairs, and (6) identification of the parent protein through a database search. This process has been tested on the selection of glycopeptides from lactoferrin and mammaglobin, and the identification of the ion pairs of fetuin glycopeptides. Glycosylation sites were identified through PNGase hydrolysis in (H2O)-O-18. During the process of hydrolyzing the conjugate, Asn is converted to an aspartate residue with the incorporation of O-18. However, PNGase F was observed to incorporate two O-18 into the P-carboxyl groups of the Asp residue. This suggests that the hydrolysis is at least partially reversible. (C) 2002 Elsevier Science B.V. All rights reserved.