Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Abstract

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.

Graphical Abstract

Highlights

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  Fractionation of membranes improves the identification of membrane proteins.

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  Membranes washed with urea or alkaline increase identified transmembrane proteins.

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  Urea wash increases the detection of multispanning transmembrane proteins.

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  Proteomics of urea-washed membranes keeps more protein–protein interactions.

In Brief

Membrane proteins, particularly integral membrane proteins, are barely detected in bottom–up proteomics because of their complex nature and abundant soluble proteins. We applied standard biochemical procedures to optimize the sample preparation method for membrane proteome. Membranes were precipitated by ultracentrifugation, followed by treatment with urea or alkaline solutions to remove contaminants. This enrichment was critical to obtain comprehensive membrane proteome data. Among the methods, washing membranes by urea distinctly revealed intricate membrane proteome with keeping protein–protein interactions.