title: 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.
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Himabindu V
authors.initials: HV
authors.lastname: Kilambi
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Kalyani
authors.initials: K
authors.lastname: Manda
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Sanivarapu
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Vineet K
authors.initials: VK
authors.lastname: Maurya
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Rameshwar
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad Hyderabad, India.
authors.forename: Yellamaraju
authors.initials: Y
authors.lastname: Sreelakshmi
keywords: protein fractionation; proteome coverage; sample preparation; shotgun proteomics; tomato fruit