Updated publication reference for PubMed record(s): 27446192. Optimization of sample preparation protocols and mass spectrometric parameters is essential for adequate proteome coverage. Though several protocols are available for 2D electrophoresis-based proteome profiling, very few protocols exist for sample preparation for shotgun proteomics and optimization of mass spectrometric parameters using plant tissues. Here, we report an optimized protocol for shotgun proteomics of tomato fruit, which is a recalcitrant tissue comprising of low concentration of proteins and high percentage of sugars and secondary metabolites