Abstract
An optimized protocol was developed for shotgun proteomics of tomato fruit, which is a recalcitrant tissue due to a high percentage of sugars and secondary metabolites. A number of protein extraction and fractionation techniques were examined for optimal protein extraction from tomato fruits followed by peptide separation on nanoLCMS. Of all evaluated extraction agents, buffer saturated phenol was the most efficient. In-gel digestion [SDS-PAGE followed by separation on LCMS (GeLCMS)] of phenol-extracted sample yielded a maximal number of proteins. For in-solution digested samples, fractionation by strong anion exchange chromatography (SAX) also gave similar high proteome coverage. For shotgun proteomic profiling, optimization of mass spectrometry parameters such as automatic gain control targets (5E+05 for MS, 1E+04 for MS/MS); ion injection times (500 ms for MS, 100 ms for MS/MS); resolution of 30,000; signal threshold of 500; top N-value of 20 and fragmentation by collision-induced dissociation yielded the highest number of proteins. Validation of the above protocol in two tomato cultivars demonstrated its reproducibility, consistency, and robustness with a CV of < 10%. The protocol facilitated the detection of five-fold higher number of proteins compared to published reports in tomato fruits. The protocol outlined would be useful for high-throughput proteome analysis from tomato fruits and can be applied to other recalcitrant tissues.
Highlights
Tomato is a good model for fleshy fruit ripening due to the availability of a high quality genome sequence, mutant collections, well characterized wild relatives, ease of transformation, etc
Plant tissues pose additional challenges owing to high level of proteases, and presence of primary and secondary metabolites that interfere with protein extraction (Saravanan and Rose, 2004)
Though several protocols are available for protein extraction (Isaacson et al, 2006; Wang et al, 2008) from fruit tissues like apple, banana (Carpentier et al, 2005; Amoako-Andoh et al, 2014), grape (Vincent et al, 2006), tomato (Saravanan and Rose, 2004) for 2DE-based proteomics, very few exist for shotgun proteomics
Summary
Tomato is a good model for fleshy fruit ripening due to the availability of a high quality genome sequence, mutant collections, well characterized wild relatives, ease of transformation, etc. It is extensively used for deciphering the molecular basis for fruit ripening at transcriptome and metabolome levels (Gapper et al, 2014; Pesaresi et al, 2014). Though several protocols are available for protein extraction (Isaacson et al, 2006; Wang et al, 2008) from fruit tissues like apple, banana (Carpentier et al, 2005; Amoako-Andoh et al, 2014), grape (Vincent et al, 2006), tomato (Saravanan and Rose, 2004) for 2DE-based proteomics, very few exist for shotgun proteomics
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