Determination of pKa values of individual histidine residues in proteins using mass spectrometry
Determination of pKa values of individual histidine residues in proteins using mass spectrometry
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DOI：
10.1021/ac8009643
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发表时间：
2008-09-01
影响因子：
7.4
通讯作者：
Nakazawa, Takashi
中科院分区：
文献类型：
--
作者：
Miyagi, Masaru;Nakazawa, Takashi
我们开发了一种质谱学方法来确定蛋白质中单个组氨酸残基的pK(A)值。该方法是基于咪唑C-2质子发生依赖于pH的氢-氚交换反应的事实，其中的速率常数k(Phi)反映了咪唑电离为咪唑的pK(A)。实验过程如下：(1)蛋白质在不同pH值的D2O溶剂中孵育；(2)用蛋白水解酶(S)消化蛋白质，在此过程中所有蛋白质快速交换。(3)用LC/ESI-MS测定每个含有组氨酸的多肽的质谱图。H-D交换反应的k(Phi)是从反映氢的掺入程度的质谱图中得到的。PK(A)值由k(Phi)与pH的关系图确定，该曲线为典型的S型曲线。根据观察到的肽的分子质量，可以同时将pK(A)值明确地分配给各个组氨酸残基。用该方法成功地测定了核糖核酸酶A中四个组氨酸残基中的三个组氨酸残基(组氨酸12、-105和-119)的pK(A)值，并与H-1核磁共振和氢氚交换法测定的结果吻合较好。该方法使用亚纳摩尔量的蛋白质，允许在比传统核磁共振方法所需的1 mM浓度低得多的浓度下进行测量，而传统核磁共振方法目前几乎是唯一的选择方法。
We developed a mass spectrometric method to determine the pK(a) values of individual histidine residues in proteins. The method is based on the fact that the imidazole C-2-proton undergoes pH-dependent hydrogen-deuterium exchange reaction, of which the rate constant (k(phi)) reflects the pK(a) for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D2O solvent at various pH values, (2) protein digestion by proteolytic enzyme(s), during which all the rapidly exchanging. deuterons such as those in amide and hydroxyl groups are back-exchanged for protons, and (3) measurement of the mass spectrum of each histidine-containing peptide by LC/ESI-MS. The k(phi) of the H-D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The pK(a) value is then determined from a plot of k(phi) versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the pK(a) values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The pK(a) values of three of four histidine residues (His12, -105, and -119) in RNase A were successfully determined by this method and were in good agreement with those determined by H-1 NMR and hydrogen-tritium exchange methods. The method uses subnanomole quantities of protein, allowing measurement at a much lower concentration than that of 1 mM required for the conventional NMR approach that is currently almost exclusively the method of choice.