Hirano Satoshi, Goto Ryohei, Uchida Yasuo
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Biomedicines. 2022 Feb 5;10(2):383. doi: 10.3390/biomedicines10020383.
The purpose of this study was to develop a method to comprehensively determine the localization of apical and basolateral membrane proteins, using a combination of apical/basolateral membrane separation and accurate SWATH (equential indow cquisition of all eoretical fragment ion spectra) proteomics. The SWATH analysis of basolateral and apical plasma membrane fractions in mouse liver quantified the protein expression of 1373 proteins. The basolateral/apical ratios of the protein expression levels were compared with the reported immunohistochemical localization for 41 model proteins (23 basolateral, 11 apical and 7 both membrane-localized proteins). Three groups were perfectly distinguished. Border lines to distinguish the apical-, both- and basolateral localizations were determined to be 0.766 and 1.42 based on probability density. The method that was established was then applied to the comprehensive determination of the proteins in mouse liver. The findings indicated that 154 and 125 proteins were localized in the apical and basolateral membranes, respectively. The levels of receptors, CD antigens and integrins, enzymes and Ras-related molecules were much higher in apical membranes than in basolateral membranes. In contrast, the levels of adhesion molecules, scaffold proteins and transporters in basolateral membranes were much higher than in apical membranes.
本研究的目的是开发一种方法，通过结合顶端/基底外侧膜分离和精确的SWATH（所有理论碎片离子光谱的顺序窗口采集）蛋白质组学，全面确定顶端和基底外侧膜蛋白的定位。对小鼠肝脏基底外侧和顶端质膜组分进行SWATH分析，对1373种蛋白质的表达进行了定量。将蛋白质表达水平的基底外侧/顶端比率与41种模型蛋白（23种基底外侧蛋白、11种顶端蛋白和7种双膜定位蛋白）的报告免疫组化定位进行了比较。三组被完美区分。根据概率密度，确定区分顶端、双膜和基底外侧定位的边界线分别为0.766和1.42。然后将建立的方法应用于小鼠肝脏蛋白质的全面测定。结果表明，分别有154种和125种蛋白质定位于顶端膜和基底外侧膜。顶端膜中受体、CD抗原和整合素、酶和Ras相关分子的水平远高于基底外侧膜。相反，基底外侧膜中粘附分子、支架蛋白和转运蛋白的水平远高于顶端膜。
