A Targeted Quantitative Proteomic Investigation Reveals the Changes in Proteins Responsible for Flavonoid and Anthocyanin Biosynthesis in Strawberry Fruit at Different Ripening Stages

To better understand the regulation of the biosynthesis of anthocyanins and other flavonoids, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars (‘Honeoye’ and ‘Mira’) at three ripeness stages. Strawberry fruit were tagged at the bloom stage and harvested at white, pink and red-ripe maturity stages. Anthocyanin concentration was positively correlated with fruit maturity with higher levels of both greater cyanidin-3-glucoside and pelargonidin-3-glucoside in riper fruit of both cultivars. A quantitative proteomic workflow was improved through OFFGEL electrophoresis to fractionate peptides obtained from total protein digests of ripening strawberry fruit. A library was established of identified proteins and peptides related to flavonoid biosynthesis from the LC/MS analysis of total protein extract from red-ripe fruit.  Our identification of proteins and peptides was improved for those of low abundance which provided per arri information. Also the current analysis provided physicochemical information about the peptides that can be used to validate the peptide sequence identification. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were examined. The normalized protein abundance measured using isotopically-labelled standards, was significantly changed greater when anthocyanin concentration was also greater in fruit of advanced maturity. The protein abundance of phenylalanine ammonia-lyase, anthocyanidin synthase, chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2 were all significantly greater in fruit of more advanced ripeness. An interaction was found between cultivar and fruit maturity with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin concentration suggested a level of metabolic control involving protein concentration. Monitoring protein levels may also reveal candidate regulatory enzymes that affect anthocyanin formation during fruit ripening.