Development of a UPLC-MRM-based targeted proteomic method to profile subcellular organelle marker proteins from human liver tissues.
Development of a UPLC-MRM-based targeted proteomic method to profile subcellular organelle marker proteins from human liver tissues.
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DOI：
10.1038/s41598-022-15171-0
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发表时间：
2022-06-29
影响因子：
4.6
通讯作者：
Wang MZ
中科院分区：
文献类型：
--
作者：
Qiu X;Doyle LM;Wang MZ
亚细胞细胞器长期以来一直是生化研究和药物开发的兴趣所在，因为分离这些细胞器有助于探索蛋白质功能和阐明药物在细胞内的处置。通常，分离的亚细胞细胞器组分的纯度是通过亚细胞细胞器标记蛋白的免疫印迹分析来确定的，这可能是劳动密集型的，并且由于抗体批次之间的差异性而缺乏重复性。因此，需要更高的吞吐量和更健壮的方法。在这里，发展了一种基于UPLC-MRM的靶向蛋白质组学方法，用于分析一组人类细胞器标记蛋白，并用于分析从人肝组织蛋白提取物中分离出的一系列蔗糖组分。通过与传统免疫印迹的比较和对与小分子和生物药物处置有关的三个案例研究蛋白(CYP3A4、FcRn和β2m)的亚细胞定位的测定，验证了该方法的有效性。所有三个案例研究蛋白都与其相应的亚细胞蛋白标记物共富集，并从分离的组份中实现了完全回收。这种新开发的用于人类细胞器标记蛋白质组的MRM方法可能会加速未来细胞内药物处置的分析，并促进亚细胞细胞器质量评估。
Subcellular organelles have long been an interest in biochemical research and drug development as the isolation of those organelles can help to probe protein functions and elucidate drug disposition within the cell. Usually, the purity of isolated subcellular organelle fractions was determined using immunoblot analysis of subcellular organelle marker proteins, which can be labor-intensive and lack reproducibility due to antibody batch-to-batch variability. As such, a higher throughput and more robust method is needed. Here, a UPLC-MRM-based targeted proteomic method was developed for a panel of human organelle marker proteins and used to profile a series of sucrose fractions isolated from the protein extract of human liver tissues. The method was validated by comparing to the traditional immunoblot and determining subcellular localization of three case study proteins (CYP3A4, FcRn, and β2M) pertaining to the disposition of small molecule and biologic drugs. All three case study proteins were co-enriched with their corresponding subcellular protein marker, and complete recoveries were achieved from isolated fractions. This newly developed MRM method for the panel of human organelle marker proteins can potentially accelerate future intracellular drug disposition analysis and facilitate subcellular organelle quality assessment.
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影响因子：
3.9
作者：
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通讯作者：
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影响因子：
64.5
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