Peptide prefractionation is essential for proteomic approaches employing multiple‐reaction monitoring of fruit proteomic research |
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| Authors: | Jun Song Leslie Campbell Palmer XiHong Li ZhaoQi Zhang |
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| Institution: | 1. Atlantic Food and Horticulture Research Centre, Agriculture and Agri‐Food Canada Kentville, Nova Scotia, Canada;2. Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin, P. R. China;3. College of Horticulture, South China Agriculture University, Guangzhou, P. R. China |
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| Abstract: | 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. |
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| Keywords: | Fruit proteomics Monodehydroascorbate reductase Quantitative proteomics Sample preparation |
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