Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages
Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages
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DOI：
10.1016/j.ijms.2008.04.030
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发表时间：
2008-12-01
影响因子：
1.8
通讯作者：
Fenselau, Catherine
中科院分区：
文献类型：
--
作者：
Li, Jinxi;Shefcheck, Kevin;Fenselau, Catherine
本实验室介绍了一种化学方法的残留物特异性蛋白质裂解，并提供了微波加速酸裂解作为蛋白质组学工具的适用性的初步评估。这份报告是一个持续评估的命运，共同的蛋白质修饰在微波加速酸裂解。我们已经检查了核糖核酸酶A和相关的N-连接的糖蛋白核糖核酸酶B，和O-连接的糖蛋白α晶体蛋白A链的切割，使用MALIDI-TOF和LC-ESI-MS来鉴定肽产物。RNA酶A和B各含有四个二硫键，并且发现需要添加还原剂如二硫苏糖醇来实现有效的酸性蛋白水解。核糖核酸酶B中的糖苷基与天冬酰胺侧链的连接被发现在12.5%乙酸中通过短暂的微波处理不被切割。将酸裂解的糖肽产物中的异质碳水化合物侧链的分布与胰蛋白酶消化的糖肽产物的分布进行比较。在所使用的条件下，碳水化合物链本身内的水解是最小的。发现α晶体A上的O-连接的侧链在蛋白质的酸裂解期间裂解。(c)2008 Elsevier B. V.保留所有权利。
This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALIDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein. (c) 2008 Elsevier B.V. All rights reserved.