A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates

Anne E. Robinson, Jeffrey P. Henderson, Katherine A. Henzler-Wildman

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.

Original languageEnglish
Pages (from-to)130-135
Number of pages6
JournalAnalytical Biochemistry
Volume549
DOIs
StatePublished - May 15 2018

Keywords

  • Mass spectrometry
  • Membrane protein
  • Membrane transport
  • Multidrug transporter
  • Transport assay

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