Derivatization for detection of abscisic acid and 12-oxo-phytodienoic acid using matrix-assisted laser desorption/ionization imaging mass spectrometry.

RATIONALE

Abscisic acid (ABA) and 12-oxo-phytodienoic acid (OPDA) play crucial roles in seed development. However, because of their low ionization efficiencies, visualization by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has been difficult. In this study, we used on-tissue chemical derivatization (OTCD) with the derivatization reagent Girard's T (GirT) in MALDI-IMS to visualize ABA and OPDA.

METHODS

Immature Phaseolus vulgaris L. seeds were homogenized, and frozen homogenate sections were prepared using a cryostat. The concentration of the trifluoroacetic acid (TFA) and spray volume of the GirT solution were optimized using the homogenate sections. Immature seed sections were prepared using a cryostat, and the OTCD efficiency under optimal conditions was measured using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The GirT solution was sprayed on the seed sections, and then MALDI-IMS was performed.

RESULTS

The optimal TFA concentration and spray volume were 2% and 500 μL, respectively. The OTCD efficiency rates were 61 ± 10% for ABA and 45 ± 5% for OPDA. The peaks corresponding to GirT-derivatized ABA (ABA-GirT) and OPDA (OPDA-GirT) standards were detected on the optimal OTCD-treated seed sections. ABA-GirT was mainly distributed in the embryo, while OPDA-GirT was localized in the external structures. These results are in agreement with our previously published results.

CONCLUSIONS

Our results show that ABA and OPDA in the immature seeds were exactly visualized using OTCD with GirT in MALDI-IMS. Therefore, OTCD with GirT in MALDI-IMS is a promising technique for future research on the biological roles of ABA and OPDA in various immature seeds.