Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics |
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Authors: | Pornparn Kongpracha Pattama Wiriyasermkul Noriyoshi Isozumi Satomi Moriyama Yoshikatsu Kanai Shushi Nagamori |
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Affiliation: | 1. Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan;2. Department of Collaborative Research for Biomolecular Dynamics, Nara Medical University, Nara, Japan;3. Department of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan |
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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. |
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Keywords: | transmembrane domain transporter urea alkaline protein complex protein–protein interaction ACN" },{" #name" :" keyword" ," $" :{" id" :" kwrd0045" }," $$" :[{" #name" :" text" ," _" :" acetonitrile AMED" },{" #name" :" keyword" ," $" :{" id" :" kwrd0055" }," $$" :[{" #name" :" text" ," _" :" Japan Agency for Medical Research and Development BCA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0075" }," $$" :[{" #name" :" text" ," _" :" bicinchoninic acid FA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0085" }," $$" :[{" #name" :" text" ," _" :" formic acid GO" },{" #name" :" keyword" ," $" :{" id" :" kwrd0095" }," $$" :[{" #name" :" text" ," _" :" Gene Ontology GRAVY" },{" #name" :" keyword" ," $" :{" id" :" kwrd0105" }," $$" :[{" #name" :" text" ," _" :" grand average of hydropathy HEK293T" },{" #name" :" keyword" ," $" :{" id" :" kwrd0115" }," $$" :[{" #name" :" text" ," _" :" human embryonic kidney 293T cell line IPA" },{" #name" :" keyword" ," $" :{" id" :" kwrd0125" }," $$" :[{" #name" :" text" ," _" :" ingenuity pathway analysis MS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0135" }," $$" :[{" #name" :" text" ," _" :" mass spectrometry PPI" },{" #name" :" keyword" ," $" :{" id" :" kwrd0145" }," $$" :[{" #name" :" text" ," _" :" protein–protein interaction PSM" },{" #name" :" keyword" ," $" :{" id" :" kwrd0155" }," $$" :[{" #name" :" text" ," _" :" peptide spectra match SDB-XC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0165" }," $$" :[{" #name" :" text" ," _" :" styrene-divinylbenzene crosslinked SDC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0175" }," $$" :[{" #name" :" text" ," _" :" sodium deoxycholate SLC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0185" }," $$" :[{" #name" :" text" ," _" :" solute carrier STRING" },{" #name" :" keyword" ," $" :{" id" :" kwrd0195" }," $$" :[{" #name" :" text" ," _" :" Search Tool for the Retrieval of Interacting Genes/Proteins TMD" },{" #name" :" keyword" ," $" :{" id" :" kwrd0205" }," $$" :[{" #name" :" text" ," _" :" transmembrane domain Tris" },{" #name" :" keyword" ," $" :{" id" :" kwrd0215" }," $$" :[{" #name" :" text" ," _" :" Tris(hydroxymethyl)aminomethane |
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