Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off-line high-pH reverse-phase phase chromatography in combination with LC-MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple-TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening.

Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using mass-spectrometry (MS) based approaches, it is now possible to routinely quantify thousands of proteins. However, pre-fractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH was introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, we built spectral libraries for two widely used models to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5,197 and 6,040 proteins for Solanum lycopersicum and Drosophila melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all massspectrometry data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495. For DIA, the spectral library contains experimental acquired spectra of the precursors and can be produced directly from the SWATH data, by reconstituting the MSMS spectra using the retention time of the precursors and the fragments [7], or by DDA analysis of the sample, which can be fractionated to increase the number of spectra in the library [9]. Ideally, the spectral library should be generated on a MS instrument similar to the one used to acquire further SWATH-MS data as the correlation of the fragment intensities for a peptide acquired on different instrument was shown to be rather low [10]. Although Rosenberger et al. recently published a library containing assays for 10,000 human proteins [9] and deep libraries for other applications such as SRM (Selected Reaction Monitoring) have been generated [11][12][13], the number of publicly available spectral libraries for SWATH-MS is still limited. Several other studies have produced libraries for other species [14][15][16]. However, spectral libraries are still missing for many species and, when available, the size of the libraries are limited and a deeper coverage of the proteome would increase the number of potential identifications in SWATH-MS analysis. In the present study, we produced spectral libraries for two well established models to study fruit ripening or animal embryogenesis, respectively Solanum lycopersicum L. and Drosophila melanogaster, for which no or low depth spectral libraries for SWATH-MS on TripleTOF instruments have been produced so far [17,18]. These libraries contain assays for more than 5,000 proteins for both species and are best suited...

No abstract

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening.