Phosphorylation of the C-terminal PECAM-1 ITIM tyrosine residue (Y₆₈₆) is required for and is independent of phosphorylation of the N-terminal PECAM-1 ITIM tyrosine residue (Y₆₆₃). MALDI-TOF mass spectra of tryptic peptides from (A) unphosphorylated, (B) phosphorylated peak 1, or (C) phosphorylated peak 2 species of PECAM_594-711Y_636,701F. Peptides A and E contain no tyrosine (Y) residues, peptide B contains only Y₆₈₆, peptides C and D contain only Y₆₆₃, and peptides F and G contain both Y₆₆₃ and Y₆₈₆ (refer to Table 2 for a complete description of the amino acid sequence and location of peptides A-G within full-length PECAM-1). Peptides denoted by a single asterisk (*) represent addition of a single phosphate, whereas those denoted by 2 asterisks (**) contain 2 phosphate moieties. Note that peptides generated from phosphorylated peak 1 include unphosphorylated species that contain either no Y residues (peptides A and E) or only Y₆₆₃ (peptides C and D) and singly phosphorylated species that contain Y₆₈₆ (peptides B*, F*, and G*). Fragments generated from phosphorylated peak 2 include unphosphorylated species that contain no Y residues (peptides A and E), singly phosphorylated species that contain either Y₆₈₆ (peptide B*) or Y₆₆₃ (peptides C* and D*), and doubly phosphorylated species that contain both Y₆₈₆ and Y₆₆₃ (peptides F** and G**). m/z indicates mass-to-charge ratio.