A New Abscisic Acid Conjugate, ABA-L-Glutamate, Determined in Different Plant Species by Combined Immunoaffinity Chromatography-Tandem Mass Spectrometry

Abstract Abscisic acid (ABA) is a phytohormone that occurs in plants at very low concentration (pmol/g fresh weight) and regulates multiple biological processes, including stomatal closure, seed germination, and responses to environmental stresses. In the present study, isolation of ABA, ABA glucosyl ester, and 11 ABA amino acid conjugates from minute quantities of plant tissue (less than 20 mg fresh weight) was achieved using a purification method based on the combination of an Oasis HLB column and an immunoaffinity sorbent. New monoclonal antibodies raised against (+)- cis,trans -ABA conjugated to BSA through its carboxyl group (C1) were characterised by enzyme-linked immunosorbent assay (ELISA) and used for immunoaffinity chromatography (IAC) gel preparation. The use of immunoaffinity purification significantly reduced matrix effects and increased the selectivity and sensitivity of subsequent UHPLC-MS/MS analysis. In addition to (+)- cis,trans -ABA and its glucosyl ester, a new abscisic acid conjugate, ABA-L-glutamate, was isolated by IAC and identified by tandem mass spectrometry in pea ( Pisum sativum L.), Lepidium sativum L. and wheat ( Triticum aestivum L.) seedlings. However, it was not found in 10-day-old seedlings of Arabidopsis thaliana or water-stressed tobacco ( Nicotiana tabacum L.) leaves. Here, the identification of an ABA conjugate with glutamic acid in plants is described for the first time.