Figure 4. CDK9 is phosphorylated on Ser175 residue in cultured cells.
(A) MS/MS analysis of recombinant CDK9. Recombinant CDK9/cyclin T1 was resolved on 10% SDS-PAGE. CDK9 was identified by Coomassie staining, in-gel digested with trypsin, and the eluted peptides were subjected to MS analysis on Thermo LTQ Orbitrap XL mass spectrometer as described in Materials and Methods. The SEQUEST search results are shown. Green, peptides identified with high probability by MS/MS analysis. Red, peptides identified with less probability. Black, peptides that were not detected. (B) purification of (32P)-labeled CDK9 for the peptide fingerprint analysis. FLAG-tagged CDK9 was expressing in 293T cells and metabolically labeled in the presence of okadaic acid. CDK9 was immunoprecipitated, resolved on 10% SDS-PAGE and stained with colloidal Coomassie (upper panel), or exposed to Phosphor imager screen lower panel. Lane 1, mock-transfected cells. Lane 2, WT CDK9. Lane 3, CDK9 S175A mutant. (C) Tryptic phosphopeptide mapping. (32P)-labeled CDK9 was trypsinized and resolved on Hunter thin layer peptide mapping electrophoresis system as described in Materials and Methods. WT CDK9 (upper panel) and CDK9 S175A (lower panel) are shown. Spots labeled as 1–3 were scraped and further analyzed by MS analysis. The results are representative from 2 experiments. (D) Base peak chromatography of Spot 1. Raw base peak chromatography data showing ion with mass 318.69 that matches to AFSLAK (M+2H)2+ peptide. Results are representative from 4 experiments. E. MS/MS spectrum of derived from Spot 3. The spectrum gives positive identification of GSQITQQSTNQSR peptide. Results are from a typical experiment of 3 performed.