Figure 4.

Identification of three phosphorylated forms of TSP9 and the corresponding phosphorylation sites. (A) The MALDI-TOF spectrum of the intact TSP9. The masses of non-, mono-, di- and triphosphorylated forms of TSP9 are indicated. (B and C) The product ion spectra obtained by positive mode electrospray ionization and collision-induced dissociation of phosphorylated peptides 8 (B) and 9 (C) (Table 1). The peptide sequences are shown in respective spectrum with the low case t designating phosphorylated threonine residues. The parent molecular ions (M+H)⁺ with m/z 687.3 (B) and (M+2H)²⁺ with m/z 498.7 (C) are indicated along with the b- (N-terminal) and y- (C-terminal) fragment ions. The fragment ions produced by neutral loss of H₃PO₄ (mass 98) are marked with asterisks. Only fragment ions that help to localize the phosphorylation sites are labeled in the spectra. (D) The product ion spectrum of the doubly charged negative ion [(M−2H)²⁻, m/z 393.7] of peptide 10 (Table 1). The characteristic fragments −79 m/z (PO), −97 m/z (H₂PO), and –708.4 m/z [(M−2H)²⁻- PO]^- are indicated. The ion with m/z –180.0 corresponds to a fragment of phospho-threonine, designated by lowercase t in the shown peptide sequence.