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首页> 外文期刊>Journal of Pharmaceutical and Biomedical Analysis: An International Journal on All Drug-Related Topics in Pharmaceutical, Biomedical and Clinical Analysis >Characterization of CYP2B6 K262R allelic variants by quantitative allele-specific proteomics using a QconCAT standard
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Characterization of CYP2B6 K262R allelic variants by quantitative allele-specific proteomics using a QconCAT standard

机译：使用QConcat标准通过定量等位基因特异性蛋白质组学表征CYP2B6 K262R等位基因变体

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Clinically-relevant proteins are routinely characterized by targeted proteomic methods, which offer high accuracy and reproducibility. However, assays developed for these techniques lack distinction between different alleles expressed in biological samples. The significance of assessing such variations in genes relevant to pharmacology will depend on their prevalence and effects on drug therapy. We propose quantitative allele-specific proteomics for simultaneous abundance measurement and determination of missense polymorphisms. We employed a targeted proteomic strategy using a QconCAT standard which included two surrogate peptides (at 1:1 ratio) for a prevalent variation of CYP2B6 (K262R) so that the two variants could be quantified directly. Measurement was carried out in 24 human liver samples, out of which 21 were genotyped. Allele-specific analysis of CYP2B6 expression was accurate and precise (CV < 9%), leading to determination of allele expression ratios (variant to wild type) for heterozygous (1.006 +/- 0.079, n=12) and homozygous (0.005 +/- 0.004, n = 8) phenotypes. The abundance of CYP2B6 was 7.4+ 7.8 pmol mg(-1) microsomal protein and showed good correlation with activity against mephenytoin (Rs = 0.91, p <0.0001; R-2 = 0.93). Comparable abundance (and activity) appeared to be associated with genotypes that express at least one wild type allele, which was corroborated by turnover values. This proof-of-principle study demonstrates the possibility of simultaneous determination of CYP2B6 phenotype and abundance by independent assessment of allele products. (C) 2019 Elsevier B.V. All rights reserved.
机译：临床相关的蛋白质通过靶向蛋白质组学方法进行常规表征，其提供高精度和再现性。然而，为这些技术开发的测定缺乏在生物样品中表达的不同等位基因之间的区别。评估与药理学相关的基因这种变化的意义将取决于它们的患病率和对药物治疗的影响。我们提出了定量等位基因特异性蛋白质组学，用于同时丰富测量和畸形多态性的测定。我们使用QConcat标准使用靶蛋白质组学策略，该标准包括两个替代肽（以1：1的比率为1：1），用于CYP2B6（K262R）的普遍变异，从而可以直接量化两个变体。测量在24例人肝脏样品中进行，其中21种被基因分型。 CYP2B6表达的等位基因特异性分析是精确的，精确的（CV <9％），导致杂合（1.006 +/- 0.079，N = 12）和纯合（0.005 + / - 0.004，n = 8）表型。 CYP2B6的丰度为7.4 + 7.8pmol Mg（-1）微粒体蛋白，与对莫夫育毒素的活性表现出良好的相关性（Rs = 0.91，P <0.0001; R-2 = 0.93）。可比较的丰度（和活性）似乎与表达至少一种野生型等位基因的基因型相关，其通过周转值证实。这种原则上的研究证明了通过对等位基因产品的独立评估来同时测定CYP2B6表型和丰富的可能性。 （c）2019 Elsevier B.v.保留所有权利。

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