TY - JOUR

T1 - Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity

AU - Jørgensen, ME

AU - Crocoll, C

AU - Halkier, BA

AU - Nour-Eldin, HH

PY - 2017

Y1 - 2017

N2 - Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides ( Jørgensen et al., 2017 ), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

AB - Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides ( Jørgensen et al., 2017 ), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

U2 - 10.21769/bioprotoc.2581

DO - 10.21769/bioprotoc.2581

M3 - Journal article

C2 - 34595263

VL - 7

JO - Bio-protocol

JF - Bio-protocol

SN - 2331-8325

IS - 20

ER -