Li Jinxi, Shefcheck Kevin, Callahan John, Fenselau Catherine
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742.
Int J Mass Spectrom. 2008 Dec 1;278(2-3):109-113. doi: 10.1016/j.ijms.2008.04.030.
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.
本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一种蛋白质组学工具的适用性进行了初步评估。本报告是对微波加速酸裂解中常见蛋白质修饰命运的持续评估。我们使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和液相色谱-电喷雾电离质谱（LC-ESI-MS）来鉴定肽产物，研究了核糖核酸酶A以及相关的N-连接糖蛋白核糖核酸酶B和O-连接糖蛋白α晶状体蛋白A链的裂解情况。核糖核酸酶A和B各自含有四个二硫键，发现需要添加一种还原试剂，如二硫苏糖醇，才能实现有效的酸性蛋白水解。发现在12.5%的乙酸中进行短暂微波处理时，核糖核酸酶B中糖苷基团与天冬酰胺侧链的连接不会被裂解。将酸裂解糖肽产物中异质碳水化合物侧链的分布与胰蛋白酶消化糖肽产物的分布进行了比较。在所使用的条件下，碳水化合物链本身的水解极少。发现在蛋白质的酸裂解过程中，α晶状体蛋白A上的O-连接侧链会被裂解。
