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 MALDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contain 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.

中文翻译：

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将微波加速的残基特异性酸裂解延伸至具有碳水化合物侧链和二硫键的蛋白质

该实验室引入了一种用于残基特异性蛋白质裂解的化学方法，并对微波加速酸裂解作为蛋白质组学工具的适用性进行了初步评估。这份报告是对微波加速酸裂解中常见蛋白质修饰的命运的持续评估。我们使用 MALDI-TOF 和 LC-ESI-MS 检测了核糖核酸酶 A 和相关 N 联糖蛋白核糖核酸酶 B 以及 O 联糖蛋白 α 晶状体蛋白 A 链的裂解，以鉴定肽产物。RNase A 和 B 各包含四个二硫键，并且发现需要添加还原剂，例如二硫苏糖醇，才能实现有效的酸性蛋白水解。发现糖苷基团与核糖核酸酶 B 中天冬酰胺侧链的连接不会通过在 12.5% 乙酸中的短暂微波处理而裂解。将异质碳水化合物侧链在酸裂解的糖肽产物中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-连接侧链在蛋白质的酸裂解过程中被裂解。