In vitro identification of kinases phosphorylating the CnaA SPRR by TiO2 enrichment and LC-MS/MS analysis.

Recombinant CnaA-AfRD protein was subjected to phosphorylation reactions using the different combination of kinases (GSK-3β, CK1, GSK-3β+CK1, CDK1, MAPK and CDK1+MAPK) and verified for phosphorylation after TiO2 enrichment and LC-MS/MS analysis. In addition, the strain expressing CnaA-EGFP was treated with GSK-3β inhibitor (GSK-3β Inhibitor VII) and the CK1 inhibitor (D4476) and the phosphorylation status of CnaA was verified by LC-MS/MS analysis. The uniquely identified phosphorylation residues or combinations of phosphorylation residues are shown. Due to the proximity of multiple phosphorylatable residues within these peptides, mass spectra from each identification were submitted to an independent algorithm (Ascore) which assigns confidences to the localization of each phosphorylation. [pS] indicates the phosphorylated residue.