Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins

Quantifying the protein levels of
drug transporters in plasma membrane
fraction helps elucidate the function of these transporters. In this
study, we conducted a proteomic evaluation of enriched drug-related
transporter proteins in plasma membrane fraction prepared from mouse
liver and kidney tissues using the membrane protein extraction kit
and a bead homogenizer. Crude and plasma membrane fractions were prepared
using either the Dounce or bead homogenizer, and protein levels were
determined using quantitative proteomics. In liver tissues, the plasma
membrane fractions were more enriched in transporter proteins than
the crude membrane fractions; the average enrichment ratios of plasma-to-crude
membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers,
respectively. The concentrations of transporter proteins in plasma
membrane fractions determined using the bead homogenizer were higher
than those determined using the Dounce homogenizer. Meanwhile, in
kidney tissues, the plasma membrane fractions were enriched in transporters
localized in the brush-border membrane to the same degree for both
the homogenizers; however, the membrane fractions obtained using either
homogenizer were not enriched in Na+/K+-ATPase
and transporters localized in the basolateral membrane. These results
indicate that fractionation, using the bead homogenizer, yielded transporter-enriched
plasma membrane fractions from mouse liver and kidney tissues; however,
no enrichment of basolateral transporters was observed in plasma membrane
fractions prepared from kidney tissues.

定量分析质膜组分(plasma membrane fraction)中的药物转运蛋白水平，有助于阐明此类转运蛋白的功能。本研究针对从小鼠肝、肾组织中提取的富集型药物相关转运蛋白，开展了蛋白质组学评估：实验采用膜蛋白提取试剂盒与珠磨式匀浆器(bead homogenizer)制备质膜组分，同时分别使用杜恩匀浆器(Dounce homogenizer)或珠磨式匀浆器制备粗膜组分与质膜组分，并通过定量蛋白质组学(quantitative proteomics)测定蛋白水平。在肝组织中，质膜组分的转运蛋白富集程度高于粗膜组分；采用杜恩匀浆器与珠磨式匀浆器时，质膜组分相较于粗膜组分的平均富集倍数分别为3.31与6.93。采用珠磨式匀浆器制备的质膜组分中，转运蛋白浓度高于杜恩匀浆器的制备产物。而在肾组织中，两种匀浆器制备的质膜组分对刷状缘膜(brush-border membrane)定位的转运蛋白的富集程度相当；但两类匀浆器制备的膜组分均未对钠钾ATP酶(Na+/K+-ATPase)与基底外侧膜(basolateral membrane)定位的转运蛋白实现富集。上述结果表明，采用珠磨式匀浆器进行组分分离，可从小鼠肝、肾组织中获得转运蛋白富集的质膜组分；但从肾组织制备的质膜组分中，未观察到基底外侧转运蛋白的富集现象。