SILAC Phospho-proteomics: Trypanosoma brucei

Pseudokinases can modulate activity of protein kinases, among other modes of influencing biological pathways. We tested a hypothesis that knockdown of Tb427tmp.160.4770 (a Pseudokinase) perturbs the Phospho-proteome of T. brucei. To discover changes in the trypanosome phospho-proteome, stable isotope labelling of amino acids in cell culture (SILAC) was performed, followed by enrichment of phosphopeptides with immobilised metal affinity chromatography (IMAC), and phospho-peptide detection/quantitation using mass spectroscopy. Trypanosomes (p2T7-Tb4770) were cultured in heavy (13C6-L-Arginine, 2H4-L-Lysine) or light isotopes (L-Arginine, L-Lysine) for SILAC. Trypanosomes grown in heavy medium were induced with tetracycline for knockdown of Tb427tmp.160.4770. Induced (Tet+, H) and uninduced (Tet-, L) samples were combined and processed together for IMAC and mass spectroscopy