Nitro-Fatty Acid Detection in Plants by High-Pressure Liquid Chromatography Coupled to Triple Quadrupole Mass...

In the last few years, the role of nitric oxide (NO) and NO-related molecules has attracted attention in the field of plant systems. In this sense, the ability of NO to mediate several posttranslational modifications (NO-PTM) in different biomolecules, such as protein tyrosine nitration or S-nitrosylation, has shown the involvement of these reactive nitrogen species in a wide range of functions in plant physiology such as the antioxidant response or the involvement in processes such as germination, growth, development, or senescence. However, growing interest has focused on the interaction of these NO-derived molecules with unsaturated fatty acids, yielding nitro-fatty acids (NO₂-FAs). It has recently been shown that these molecules are involved in key signaling pathways in animal systems through the implementation of antioxidant and anti-inflammatory responses. Nevertheless, this interaction has been poorly studied in plant systems. Very recently, the endogenous presence of NO₂-FAs in the model plant Arabidopsis thaliana has been demonstrated as well as the significant involvement of nitro-linolenic acid (NO₂-Ln) in the defence response against several abiotic and oxidative stress conditions. In this respect, the detection of NO₂-FAs in plant systems can be a useful tool to determine the importance of these molecules in the regulation of different biochemical pathways. Using high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), the methods described in this chapter enable the determination of the NO₂-FA content in a pM range as well as the characterization of these nitrated derivatives of unsaturated fatty acids in plant tissues.