Department of Biological and Environmental Science, Texas A&M University at Commerce, Commerce, TX 75429, USA.
J Inorg Biochem. 2011 May;105(5):675-83. doi: 10.1016/j.jinorgbio.2011.01.010. Epub 2011 Jan 26.
Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-Pd(en)(H(2)O)(2) (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0%wt./vol. of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 at 2.5<pH<2.9 and 55-60°C for 3-72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.
用于特异性蛋白质切割的最常用试剂是蛋白酶。由于蛋白酶会被去污剂变性和失活，因此对于需要去污剂作为增溶剂的疏水性蛋白质的蛋白质组学分析来说，它们并不方便。我们使用顺式-Pd(en)(H(2)O)(2)（其中 en 代表乙二胺）作为人工蛋白酶，分别在三个牛蛋白（即泛素、β-酪蛋白和血清白蛋白）的独立实验中进行切割。在 pH 值为 2.5<pH<2.9 且温度为 55-60°C 的条件下，在含有 1.0%wt./vol. 3-[(3-胆酰胺丙基)二甲氨基]-1-丙磺酸钠（CHAPS）或 Zwittergent 3-14 的水溶液中进行切割，时间为 3-72 小时。通过 HPLC 分离消化产物，并通过串联质谱分析。通过从头测序鉴定肽段，并与牛基因组进行匹配。由于钯（II）配合物的切割相当具有选择性，因此并不常见，消化产物中 72%的鉴定肽段含有超过 10 个氨基酸。钯（II）配合物有望成为膜蛋白蛋白质组学研究中的切割试剂。
