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Development of liquid chromatography/mass spectrometry based screening assay for Pf TrxR inhibitors using relative quantitation of intact thioredoxin
Author(s) -
Munigunti Ranjith,
Calderón Angela I.
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6316
Subject(s) - chemistry , chromatography , thioredoxin reductase , mass spectrometry , detection limit , selected reaction monitoring , electrospray ionization , liquid chromatography–mass spectrometry , thioredoxin , enzyme , tandem mass spectrometry , biochemistry
RATIONALE Plasmodium falciparum ( Pf ) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx–S 2 ) to thioredoxin dithiol (Trx–(SH) 2 ) that is essential for antioxidant defense mechanism and DNA synthesis in the parasite and is a validated drug target for new antimalarial agents. METHODS In this study, we have developed a liquid chromatography/mass spectrometry (LC/MS)‐based functional assay to identify inhibitors of Pf TrxR by quantifying the product formed (Trx–(SH) 2 ) in the enzymatic reaction. Relative quantitation of the reaction product (intact Trx–(SH) 2 ) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with a positive mode electrospray ionization (ESI) source. RESULTS The calibration curve prepared for Trx–(SH) 2 at concentrations ranging from 1.8 to 116.5 µg/mL was linear (R 2 >0.998). The limit of detection (LOD) and limit of quantification (LOQ) of Trx–(SH) 2 were at 0.45 and 1.8 µg/mL respectively. To validate the developed functional assay we have screened reference compounds 1 , 2 and 3 for their Pf TrxR inhibitory activity and ten natural compounds (at 10 μM) which were earlier identified as ligands of Pf TrxR by a UF‐LC/MS based binding assay. CONCLUSIONS The developed LC/MS‐based functional assay for identification of inhibitors of Pf TrxR is a sensitive and reliable method that is also amendable for high‐throughput format. This is the first representation of a relative quantitation of intact Trx–(SH) 2 using LC/MS. Copyright © 2012 John Wiley & Sons, Ltd.