Sterol and Brassinosteroid Hormone Quantification by LC/MS of Picolinyl Ester Derivatives

Brassinosteroids are small steroidal hormones that regulate plant growth, differentiation, and defense. They are low abundance in plant tissues and are difficult to assess via mass spectrometry due to poor ionization. In this protocol, we provide a method for the extraction, detection, and quantification of a subset of sterol and brassinosteroid metabolites using a derivatization method to improve ionization during liquid chromatography coupled with mass spectrometry. Multiple reaction monitoring, which is the utilization of metabolite fragments made in the instrument, is used to distinguish the sterols from other metabolites in complex mixtures to allow the simultaneous detection of a wide variety of steroids, including brassinosteroids. In maize, genetic resources have permitted multiple insights into the role of brassinosteroids in growth and development, and the addition of this convenient protocol to quantify their levels in plant tissue will enable a deeper physiological and biochemical understanding.