Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.
Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.
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DOI：
10.1021/acs.analchem.8b01913
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发表时间：
2018-10-16
影响因子：
7.4
通讯作者：
Prasad B
中科院分区：
文献类型：
--
作者：
Xu M;Saxena N;Vrana M;Zhang H;Kumar V;Billington S;Khojasteh C;Heyward S;Unadkat JD;Prasad B
组织匀浆的亚细胞分离提供了丰富的体外模型（例如，微粒体，细胞质或膜），这些模型通常用于药物代谢或转运蛋白活性和蛋白质丰度研究。然而，亚细胞组分的回收、富集和纯度的批次间或实验室间的差异会影响体外模型的性能，导致药物清除的体外到体内外推（IVIVE）不准确。为了评估亚细胞组分的质量，我们开发了一种简单的靶向和敏感的LC-MS/MS蛋白质组学策略，该策略依赖于测定各种细胞器的蛋白质标志物，即质膜、细胞质、细胞核、线粒体、内质网（ER）、溶酶体、过氧化物酶体、细胞骨架和外泌体。定量蛋白质组学方法的应用证实了处理变量（即均质方法和离心速度）对微粒体和细胞质溶胶中分离蛋白的回收、富集和纯度有显著影响。特别是，内质网管腔和线粒体管腔的标记物在细胞质组分中富集，这是它们在均质过程中释放的结果。同样，从细胞和组织样品中分离的总膜组分的相对回收率和组成在定量上是不同的，在IVIVE中应予以考虑。此外，对从三明治培养肝细胞培养基中分离的外泌体的分析显示，培养时间对纯化外泌体的组成有影响。因此，与基于抗体或活性的分析相比，本文开发的基于定量蛋白质组学的策略可用于有效和同时测定各种细胞器的多种蛋白质标记物，并可用于亚细胞分离过程的质量控制，包括用于药物代谢和转运研究的体外模型开发。
Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or inter-laboratory variability in the recovery, enrichment and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple targeted and sensitive LC-MS/MS proteomics based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs. tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays, and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.
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影响因子：
82.9
作者：
通讯作者：
--
影响因子：
56.9
作者：
Bonifati, V;Rizzu, P;Heutink, P
通讯作者：
Heutink, P
影响因子：
3.9
作者：
Cubitt, Helen E.;Houston, J. Brian;Galetin, Aleksandra
通讯作者：
Galetin, Aleksandra
DOI：
10.1007/bf03160118
发表时间：
1993-04-01
期刊：
MOLECULAR AND CHEMICAL NEUROPATHOLOGY
影响因子：
--
作者：
FAFFMICHALAK, L;ALBRECHT, J
通讯作者：
ALBRECHT, J
影响因子：
4.5
作者：
Kumar, Vineet;Tot Bui Nguyen;Unadkat, Jashvant D.
通讯作者：
Unadkat, Jashvant D.