Implementation of a LC-MS/MS Method for the Detection and Quantification of Multiple Plant

Implementation of a LC-MS/MS method for the detection and quantification of multiple plant hormones

Scientific advisers: Carla Pinheiro (Plant Biochemistry) and Ana Varela Coelho (Mass Spectrometry)

Duration: 9 months

Local: ITQB (Oeiras)

The hormonal balance (achieved through the coordination of local and long distance signals) provides a mechanism linking environmental cues with biological responses, and with plant fitness and survival. The ability to measure hormones quantitatively is therefore biologically important. Traditionally, plant hormones are quantified through commercially-available competitive ELISA immunoassays but this methodology has several drawbacks. More recently, liquid chromatography-mass spectrometry (LC-MS/MS) based methods have been adopted for the quantification of multiple hormones in a simpler, faster, sensitive, reproducible and automated way. This methodology also allows greater selectivity (1,2). We propose to implement and validate, at ITQB, plant hormone quantification using LC-MS/MS.

We will focus on 3 major hormone groups (ABA, IAA and bioactive cytokinins) for the optimization of the LC-MS/MS procedure: HPLC running conditions; detection of product ion; limits of detection (LOD); limits of quantitation (LOQ); recovery ratios and quantitative precision. The quantitative analyses of the hormones will be made by multiple reaction monitoring (MRM) of the major fragment ions of the analyte. The LC-MS/MS quantification method will be implemented using the HPLC-MS systems available, a linear ion trap mass spectrometer (LTQ, Thermo-Finnigan) and a tri-dimensional ion trap mass spectrometer (LCQ, Thermo-Finnigan), both with electrospray ionization and coupled to RP HPLC (Thermo-Finnigan). Automatic data acquisition and processing will be performed using the Xcalibur software package (Thermo-Finnigan), using the procedures currently in use at the ITQB Mass Spectrometry Laboratory.

ABA, IAA, bioactive Cks will be extracted according Pan et al. (1) and quantified in plant biological samples. The effects of slowly imposed soil water deficit on the several organs of Lupinusalbus will be evaluated in what concerns hormonal balance and plant performance (3).

References

1)Pan, X. Q., Welti, R. & Wang, X. M. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nature Protocols 5, 986-992, doi:10.1038/nprot.2010.37 (2010)

2)Albacete, A. et al. Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanumlycopersicum L.) plants. Journal of Experimental Botany 59, 4119-4131, doi:10.1093/jxb/ern251 (2008).

3)Pinheiro,C., António, C., Ortuño, M.F., Dobrev, P.I., Hartung, W., Thomas-Oates, J., Ricardo, C.P., Vanková, R., Wilson, J.C. & Chaves, M.M.Initial water deficit effects on Lupinusalbus photosynthetic performance, carbon metabolism and hormonal balance: metabolic reorganisation prior to early stress responses (submitted).