Table 1.

Selected peptides from native afadin (uniprot: F1PSU6) identified by LC-MS/MS

Position	Afadin Peptide (Dog)	m/z	CS/LCM Normalized Ratio (n=6)	CS/LCM Ratio 95% CI	Putative Kinase
S228	226-TISNPEVVMK-235	1196.551	1.703	1.399–2.007	aPKC
S1102	1100-TSSVVTLEVAK-1110	1,212.6	0.9413	0.8695–1.013	AMPK

Confluent wild-type (WT) Madin-Darby canine kidney (MDCK) cells were incubated in media containing 5 μM Ca²⁺ (LCM) for 16 h. Cells undergoing the Ca²⁺ switch (CS) were introduced to media containing 1.8 mM Ca²⁺ for 2 h. Phosphopeptides were identified through LC-MS/MS analysis of afadin immunoprecipitated from MDCK cells subjected to LCM or Ca²⁺ switch conditions. Afadin peptides were subjected to titanium dioxide (TiO₂) enrichment to isolate phosphopeptides. Unambiguous assignment of phosphorylation sites is indicated by S. Normalized ratios were obtained by comparing the phosphopeptide ratios of TiO₂-enriched samples relative to the ratios of their nonphosphorylated counterparts (n = 6). Putative kinases were determined by searching MIT Scansite 4.0. aPKC, atypical protein complex C; CI, confidence interval; LCM, low-Ca²⁺ media.