Alkaline cleavage of covalent bonds in chicken insulin and bovine α‐lactalbumin analyzed by matrix‐assisted laser desorption/ionization‐ mass spectrometry
Alkaline cleavage of covalent bonds in chicken insulin and bovine α‐lactalbumin analyzed by matrix‐assisted laser desorption/ionization‐ mass spectrometry
复制标题
DOI：
10.1002/elps.200406153
复制
发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
T. Manabe;Ya Jin
中科院分区：
文献类型：
--
作者：
T. Manabe;Ya Jin
在寻找从考马斯蓝染色聚丙烯酰胺凝胶中提取蛋白质的方法过程中，我们发现当凝胶片浸泡在碱性溶液中时，蛋白质的提取回收率相对较高。然而，已知碱性条件会导致蛋白质分解，尤其是肽键断裂和二硫键降解。我们研究了碱性对两种纯化蛋白质（鸡胰岛素和牛 α-乳清蛋白）的影响，这两种蛋白质的结构中都含有四个二硫键。通过使用基质辅助激光解吸/电离质谱（MALDI-MS）分析蛋白质的质谱来追踪共价键裂解的过程。当蛋白质在 0.1% 二硫苏糖醇 (DTT) 存在下保持在 pH 13 时，天冬酰胺酰残基 C 末端的肽键优选被裂解，产生琥珀酰亚胺，而半胱氨酰残基不会分解。在没有DTT的情况下，蛋白质的二硫键被碱分解，肽键的断裂不太明显，可能是因为蛋白质的构象被部分保留，直到二硫键完全分解。这些结果首次确定了碱处理下蛋白质的裂解位点，并进一步表明了在存在和不存在 DTT 的情况下反应的总体趋势。
In the course of searching methods to extract proteins from Coomassie blue‐stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine α‐lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C‐terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.