TY - JOUR
T1 - A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates
AU - Robinson, Anne E.
AU - Henderson, Jeffrey P.
AU - Henzler-Wildman, Katherine A.
N1 - Funding Information:
We thank Geoff Chang for the EmrE expression plasmid and Shannon Ohlemacher and Robin Shields-Cutler for helpful conversations about the MS transport assay. This work was supported in whole or part by the National Institutes of Health ( 1R01GM095839 to K.A.H-W. and RO1DK099534 to J.P.H.), an NSF graduate research fellowship to A.R. ( DGE-1143954 ), a Mr. and Mrs. Spencer T. Olin Fellowship for Women in Graduate Study to A.R., and a Career Award from the Burroughs Wellcome Fund to J.P.H.
Publisher Copyright:
© 2018 Elsevier Inc.
PY - 2018/5/15
Y1 - 2018/5/15
N2 - 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.
AB - 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.
KW - Mass spectrometry
KW - Membrane protein
KW - Membrane transport
KW - Multidrug transporter
KW - Transport assay
UR - http://www.scopus.com/inward/record.url?scp=85044474884&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2018.03.017
DO - 10.1016/j.ab.2018.03.017
M3 - Article
C2 - 29559333
AN - SCOPUS:85044474884
SN - 0003-2697
VL - 549
SP - 130
EP - 135
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -