Ion chromatogram of KG1a cells before and after coculture with HS5 stroma. Mass spectra of noncocultured (top panel) and cocultured (bottom panel) KG1a cells at 4-hour time points both spectra filtered for PARK-7 peptide sequence ALVILA at a mass to charge ratio of 364.5. KG1a cell lysates were trypsin digested, solid-phase extraction purified, and exposed to phospho-Tyr-100 immunoaffinity beads as described in “Cell lysates.” The phosphopeptide-enriched samples were dried, resuspended, and analyzed by LC-MS. Tandem mass spectra were searched against a human International Protein Index database by use of the X!Tandem protein search engine and evaluated by the Bayesian statistical algorithm, PeptideProphet. Two unique peptides for PARK-7 were identified in the noncocultured assay; the peptide with sequence ALVILA (amino acid sequence) and mass to charge ratio of 364.5 eluted at 28.1 minutes in the filtered ion chromatogram (X). The ion peak for ALVILA was not observed in filtered ion chromatograms for the 30-minute, 1-hour, 4-hour, and 24-hour cocultured time points (X through X; shown is the 4-hour time point); the database search identified no PARK-7 peptides in these samples (see supplemental Figure 1).