SWATH-Based Comprehensive Determination of the Localization of Apical and Basolateral Membrane Proteins Using Mouse Liver as a Model Tissue.

Satoshi Hirano,Ryohei Goto,Yasuo Uchida

doi:10.3390/biomedicines10020383

Abstract

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 (Sequential Window Acquisition of all THeoretical 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.

Highlights

SWATH analysis of basolateral and apical plasma membrane fractions in mouse liver showed that 1373 proteins were quantified (Table S2)
To avoid the inclusion of molecules that are localized to organelle membranes in the cell, we focused on molecules that have been shown to be localized to cell membranes in the uniprot database
It is known that asialoglycoprotein receptor (Asgr) is localized on basolateral membranes, in this study, we were able to separately quantify Asgr1 and Asgr2, and the results clearly shows that both molecules are localized on basolateral membranes (Table 2)

Summary

Introduction

Tissues harbor two types of cell membranes, namely, apical and basolateral membranes. The membrane on the opposite side of the basolateral membrane in polarized cells is referred to as the “apical membrane”. Membrane proteins that are localized on the blood side of the cell membrane need to be targeted. In order to achieve this, it is important to have available a comprehensive list of membrane proteins that are localized on apical and basolateral cell membranes in each organ. This task is best suited to comprehensive profiling approaches such as proteomics, rather than laborious immunohistochemical analysis

Objectives

Methods

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Discussion

Conclusion