The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis.
The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis.
复制标题
金属硫蛋白对蛋白水解消化的抗性：LC-MS/MS 分析。
DOI：
10.1002/elps.200600835
复制
发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Lu,Xiaoning
中科院分区：
文献类型：
--
作者：
Wang,Rongying;Sens,DonaldA;Garrett,Scott;Somji,Seema;Sens,MaryAnn;Lu,Xiaoning
金属硫蛋白（MT）是富含半胱氨酸的金属蛋白家族，与重金属如Cd（II），Zn（II）和Cu（I）强烈结合。其他研究小组使用凝胶电泳和荧光的先前工作表明，MT对多种酶的蛋白水解消化具有抗性，这增加了MT蛋白质组学鉴定的难度。本工作试图使用LC-MS/MS（LC-MS/MS）分析MT对胰蛋白酶的抗性，其能够确定产生的肽的序列，从而精确地表征切割。结果表明，金属饱和的MT对胰蛋白酶完全耐受。这种抗性问题可以通过向MT样品中加入EDTA来克服，这使得MT容易消化成肽并通过MS/MS鉴定。有趣的是，部分金属结合的MT被消化成肽，主要是与错过裂解，这很好地依赖于与MT结合的重金属的量。对这些意见提出了解释。证明了MT对胰蛋白酶的抗性在复杂混合物如培养细胞中MT的分离和鉴定中的潜在应用。初步数据还显示，相同的蛋白质组学方法的蛋白水解消化，然后进行MS/MS分析，可以提供信息的金属结合状态的MT，沿着鉴定的MT混合物。
Metallothioneins (MTs) are a family of cysteine‐rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC‐MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal‐saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.