Peptide prefractionation is essential for proteomic approaches employing multiple-reaction monitoring of fruit proteomic research.

Off-gel™ IEF has become a popular tool in proteomics research to fractionate peptides or proteins. We conducted a detailed investigation on the fruit proteomics of apple, banana, and strawberry fruit employing Off-gel™ electrophoresis (OGE) as a crucial step to improve the proteome coverage and quantitative proteomic workflows including multiple-reaction monitoring (MRM). We provide technical details concerning the application of Off-gel™IEF, nano-LC-MS detection, and MRM optimization and analysis. Our results demonstrated that the application of OGE is an effective method for peptide fractionation and increased significantly the number of proteins identified by at least ten times, with more total peptides detected and collected. Furthermore, we developed a protocol combining OGE and MRM studies to identify and quantitatively investigate monodehydroascorbate reductase, a key enzyme in the redox and antioxidant system of apple fruit during fruit ripening. Using this method, the quantitative changes in this protein during ripening and in response to ethylene treatment was investigated. Our results provide direct and comprehensive evidence demonstrating the benefits of OGE and its application for both shotgun and quantitative proteomics research.