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 t

SUBMITTER: Kilambi HV 

PROVIDER: S-EPMC4925719 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature