Identification and quantification of the ethylene signalling components in tomato through targeted proteomics

Ripening of climacteric fruit is directly controlled by the hormone ethylene. Understanding its biosynthesis and perception on the fruit level can lead us to optimize better ways to handle and preserve fruit after harvest. The perception of ethylene is performed by the ethylene receptors, which are the first step in the complex pathway of the ethylene signal transduction. The studies on the ethylene signalling have advanced considerably in revealing the functioning of the complex pathway, but there is still a lot of work to be done in the proteomic characterization of the main proteins involved to understand their regulation, for instance, during ripening. This work focuses on the protein identification and subsequent quantification of the ethylene receptors (ETRs), and CONSTITUTIVE TRIPLE RESPONSES (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2), next downstream components of the pathway, in tomato, a model fruit to study fruit ripening. The low abundance of these membrane proteins made this work very challenging, necessitating sample fractionation and mass spectrometry analysis with the targeted protein technique Parallel Reaction Monitoring to identify them. As a result, seven ETRs, three CTRs and EIN2 have been identified and quantified with control labelled peptides enriched with heavy stable isotopes as internal standards. This work shows the feasibility of the identification and quantification of the main proteins of the ethylene signal transduction in tomato to understand their turnover during climacteric fruit ripening.