title: Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry.
abstract: A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.
authors.affiliation: Cargill Central Research, Minneapolis, MN 55440-5702, USA. joseph_dalluge@cargill.com
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Dalluge
authors.forename: Steven
authors.initials: S
authors.lastname: Gort
authors.forename: Russell
authors.initials: R
authors.lastname: Hobson
authors.forename: Olga
authors.initials: O
authors.lastname: Selifonova
authors.forename: Frank
authors.initials: F
authors.lastname: Amore
authors.forename: Ravi
authors.initials: R
authors.lastname: Gokarn
mesh_terms: D002138:Calibration; D002264:Carboxylic Acids / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D002264:Carboxylic Acids; C036820:coenzyme A thiosulfonate; D003065:Coenzyme A