Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

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.

Label‐free deep shotgun proteomics reveals protein dynamics during tomato fruit tissues development

“…Finally, a recent method optimization study by Kilambi et al . () reported the identification of 5404 tomato fruit proteins using sample pre‐fractionation by in‐gel peptide isoelectric focusing and a nano‐LC coupled with high‐resolution mass spectrometry (HRMS). However, of all identified proteins, only 2664 could be quantified by spectral counting.…”

Comparative analysis of seed proteome of Glycine max and Glycine soja

“…In the present study, we investigated the seed proteome of G. max and G. soja by a label‐free quantitative technique combined with PSP to elucidate the differences in low‐abundant proteins. Gel‐free label‐free quantitative proteomics (Neilson et al., 2011) has been conducted in crops including soybean (Kamal, Rashid, Sakata, & Komatsu, 2015; Nouri & Komatsu, 2010), tomato ( Solanum lycopersicum L.; Kilambi et al., 2016), barley ( Hordeum vulgare L.; Mahalingam, 2017), rice ( Oryza sativa L.; Neilson et al., 2011), and corn ( Zea mays L.; Marcon et al., 2013). The difficulty in seed proteomics is that the abundant seed storage proteins have often hampered deep coverage of the seed proteome.…”

In‐depth characterization of the tomato fruit pericarp proteome

“…Ultimately, the genome, proteome, and metabolome must be fully analyzed and integrated to completely understand the phenotype of the fruit. To date, the most recent high-throughput proteomics analyses were reported by Martin et al [5] and Kilambi et al [6], who identified 3625 and 5404 proteins, respectively. Zhang et al [7] recently reported the identification of more than 10 000 proteins from tomato leaves with a global FDR of 5%.…”