Determination of δ-[L-α-aminoadipyl]-L-cysteinyl-D-valine in cell extracts of Penicillium chrysogenum using ion pair-RP-UPLC-MS/MS.
δ-[L-α-Aminoadipyl]-L-cysteinyl-D-valine (ACV) is a key intermediate in the biosynthesis pathway of penicillins and cephalosporins. Therefore, the accurate quantification of ACV is relevant, e.g. for kinetic studies on the production of these β-lactam antibiotics. However, accurate quantification of ACV is a challenge, because it is an active thiol compound which, upon exposure to air, can easily react with other thiol compounds to form oxidized disulfides. We have found that, during exposure to air, the oxidation of ACV occurs both in aqueous standard solutions as well as in biological samples. Qualitative and quantitative determinations of ACV and the oxidized dimer bis-δ-[L-α-aminoadipyl]-L-cysteinyl-D-valine have been carried out using ion pair reversed-phase ultra high-performance liquid chromatography, hyphenated with tandem mass spectrometry (IP-RP-UPLC-MS/MS) as the analytical platform. We show that by application of tris(2-carboxy-ethyl)phosphine hydrochloride (TCEP) as the reducing reagent, the total amount of ACV can be determined, while using maleimide as derivatizing reagent enables to quantify the free reduced form only.
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. r.malekiseifar@tudelft.nl
Reza Maleki
RM
Seifar
Amit T
AT
Deshmukh
Joseph J
JJ
Heijnen
Walter M
WM
van Gulik
D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D007202:Indicators and Reagents; D008301:Maleimides; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010408:Penicillium chrysogenum / Q000737:chemistry* / Q000378:metabolism; D010720:Phosphines; D053719:Tandem Mass Spectrometry / Q000379:methods*; D047090:beta-Lactams / Q000378:metabolism
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D008301:Maleimides; D009842:Oligopeptides; D010720:Phosphines; D047090:beta-Lactams; C011638:5-(2-aminoadipyl)cysteinylvaline; C080938:tris(2-carboxyethyl)phosphine; C043592:maleimide
Cell extract; Maleimide; Penicillium chrysogenum; TCEP; Thiols