Molecular Structure Analysis, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, Germany.
Anal Chem. 2010 Mar 15;82(6):2334-40. doi: 10.1021/ac9025412.
A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications.
描述了一种新颖的、广泛适用的绝对定量蛋白质生产方法，该方法可作为基于质谱的定量蛋白质组学研究的内标。这些标准是含有同位素标记和硒代甲硫氨酸的重组蛋白。为了进行重组蛋白表达，使用 gateway 技术组装适合无细胞蛋白合成的表达载体，该技术通过开放阅读框文库提供了快速访问各种基因的途径，并且易于在载体之间穿梭基因。通过无细胞表达在甲硫氨酸被硒代甲硫氨酸取代且至少一个氨基酸被高度稳定的同位素标记类似物取代的培养基中生成蛋白质。在蛋白质合成和纯化后，使用元素质谱法对硒进行绝对定量，而蛋白质中的重氨基酸则在随后的 LC-ESI-MS 或 MALDI-MS 分析中作为参考。因此，这些标准被命名为 RISQ（用于重组同位素标记和硒定量）蛋白。在这项研究中，生成了一种含有 Lys+6([(13)C(6)]-赖氨酸)和 Arg+10([(13)C(6),(15)N(4)]-精氨酸)的蛋白质，使得每个标准胰蛋白酶肽都含有一个标记的氨基酸。载脂蛋白 A1 被合成作为 RISQ 蛋白，其作为内标用于定量参考物质在指定值内。由于它们的无细胞表达，RISQ 蛋白不包含翻译后修饰。因此，ESI 或 MALDI-MS 的正确定量数据仅限于基于分析物蛋白未修饰区域衍生的肽进行的定量。因此，RISQ 蛋白除了作为内标外，还是分析共价蛋白质修饰的新工具。
