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Recombinant Isotope Labeled and Selenium Quantified Proteins for Absolute Protein Quantification

机译:重组同位素标记和硒定量蛋白质用于绝对蛋白质定量

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摘要

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 Al 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 post-translational 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.
机译:描述了一种生产绝对定量蛋白质的新颖,广泛应用的方法,该方法可用作基于质谱的定量蛋白质组学研究的内部标准。这些标准品是含有同位素标记和硒代蛋氨酸的重组蛋白。对于重组蛋白表达,使用网关技术进行了适合无细胞蛋白合成的表达载体的组装,该技术可通过开放阅读框文库快速访问多种基因,并且载体之间的基因易于穿梭。这些蛋白质是通过在无蛋氨酸的培养基中无细胞表达而产生的,在该培养基中,蛋氨酸与硒代蛋氨酸交换,而至少一个氨基酸被高度稳定的同位素标记的类似物交换。蛋白质合成和纯化后,硒通过元素质谱法用于绝对定量,而蛋白质中的重氨基酸在随后的LC-ESI-MS或MALDI-MS分析中用作参考。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,生成了包含Lys + 6([〜(13)C_(6)]-赖氨酸)和Arg + 10([〜(13)C_(6),〜(15)N_(4)]的蛋白质。 -精氨酸),以便每个标准胰蛋白酶肽都包含一个标记的氨基酸。载脂蛋白A1被合成为RISQ蛋白,其作为内标的使用导致在指定值内对参考物质进行定量。由于其无细胞表达,RISQ蛋白不包含翻译后修饰。因此,通过ESI-或MALDI-MS得出的正确定量数据仅限于基于衍生自分析物蛋白质未修饰区域的肽进行的定量。因此,除了作为内标外,RISQ蛋白还可以作为定量分析共价蛋白修饰的新工具。

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