A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry.
A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry.
复制标题
用于定量蛋白质甲基化化学计量的液相色谱串联质谱方法。
DOI：
10.1016/j.ab.2018.01.018
复制
发表时间：
2018
影响因子：
2.9
通讯作者：
Kuret,Jeff
中科院分区：
文献类型：
--
作者：
Cooper,GraceL;Huseby,CarolJ;Chandler,ClaireN;Cocuron,Jean-Christophe;Alonso,AnaP;Kuret,Jeff
翻译后修饰是生物学上重要且广泛的蛋白质功能调节剂。虽然用于检测特定修饰的存在的方法正在建立，但用于定量其摩尔修饰/摩尔蛋白质化学计量的方法还不太发达。在这里，我们介绍了一个比例，无标记，有针对性的液相色谱串联质谱为基础的方法，估计赖氨酸和精氨酸甲基化后修饰的蛋白质的化学计量。甲基化的赖氨酸和精氨酸检测定量限在低fmol和线性范围从20至5000 fmol。这种灵敏度水平允许从微克量的各种蛋白质（包括来自重组或组织来源的那些蛋白质）估计甲基化化学计量。该方法还将总甲基化化学计量分解成其基本的单甲基化、二甲基化和三甲基化残基组分。除了与蛋白质甲基化的动力学实验兼容外，该方法对于表征从细胞和组织中分离的蛋白质的甲基化状态特别有用。
Post-translational modifications are biologically important and wide-spread modulators of protein function. Although methods for detecting the presence of specific modifications are becoming established, approaches for quantifying their mol modification/mol protein stoichiometry are less well developed. Here we introduce a ratiometric, label-free, targeted liquid chromatography tandem mass spectroscopy-based method for estimating Lys and Arg methylation stoichiometry on post-translationally modified proteins. Methylated Lys and Arg were detected with limits of quantification at low fmol and with linearity extending from 20 to 5000 fmol. This level of sensitivity allowed estimation of methylation stoichiometry from microgram quantities of various proteins, including those derived from either recombinant or tissue sources. The method also disaggregated total methylation stoichiometry into its elementary mono-, di-, and tri-methylated residue components. In addition to being compatible with kinetic experiments of protein methylation, the approach will be especially useful for characterizing methylation states of proteins isolated from cells and tissues.