Abstract: 【Objective】Apple skin color is an important fruit quality factor. In order to make clear whether there is phosphorylation during apple fruit pigmentation, large scale identification of phosphoproteins and phosphopeptides was conducted. 【Method】The fruit peels of red skinned apple cultivar ‘Gala’ and non-red skinned apple cultivar ‘Golden Delicious’ were used to extract protein. Fruit protein was digested with trypsin, the resulting peptides were subjected to a step of TiO₂ beads enrichment to obtain phosphopeptides, and a step of HPLC separation．Q-Exactive mass spectrometer was used to identify phosphopeptides. The software Mascot 2.2 and Proteome Discoverer1.4 (thermo) were used to identify proteins in the database Uniprot_Maloideae.fasta. Then data analysis was performed to provide information of identified phosphorylation sites in red and non-red apple cultivars．【Result】 1 323 proteins, 3 339 peptides, 225 phosphopeptides were identified and about 200-225 phosphoproteins were found in the red apple cultivars, 569 proteins, 1 152 peptides, 128 phosphopeptides were identified and about 117-128 phosphoproteins were found in the non-red apple cultivars．Comparative analysis indicated that 43 phosphopeptides were only found in non-red apple cultivars, 139 phosphopeptides were only discovered in red apple cultivars, which proved that more phosphoproteins produced in red apple cultivars, including plasma membrane H⁺-ATPase with two phosphorylation sites in serine, a major allergen and two protein kinases with phosphorylation site in threonine, three transcription factors, a potassium transporter and a putative protein receptor kinase. Among the identified proteins in the red and non-red apple cultivars, the phosphorylation sites were mainly found in threonine, some in serine, seldom in tyrosine. There was no phosphorylation site in the C4H, 4CL, CHS, CHI, F3′H, FLS, LDOX and UFGT proteins of anthocyanin synthesis pathway and protein WD40, the MYBR transcription factor with phosphorylation site was found in both red and non-red apple cultivars. 【Conclusion】 Some phosphoproteins only produced in red apple cultivars, including plasma membrane H⁺-ATPase related to apple pigmentation. Apple fruit pigmentation is possibly influenced by protein phosphorylation.

Key words: apple, phosphorylation, proteome, protein kinase, pigmentation