Serine 37 or Serine 38 of CTMP is phosphorylated in vivo following stimulation of CCL64 cells with pervanadate. CCL64 cells expressing Flag-CTMP were metabolically labeled with 32Pi prior to pervanadate treatment. (A) Immunoprecipitated 32P-labeled CTMP and (B) Extracted protein from both conditions was hydrolyzed and analyzed as described in Experimental Procedures. (Phosphoserine: pS, phosphothreonine: pT, and phosphotyrosine: pY) (C) Full scan mass spectrum of fraction 16 in the positive mode was acquired. (D) m/z values of the phosphopeptides were identified by m/z 79 precursor-ion scanning in the negative ion mode. (E and F). Tandem mass spectra derived by CID of the (M + 2H) 2+ precursor, m/z 609.3. Fragment ions in the spectra represent many single-event preferential cleavages of peptide bonds, resulting in the sequence information recorded from both the N- and C-termini of the peptide simultaneously. The single-letter code for the amino acids is shown.