SILAC-based quantitation of changes in the phosphorylation of invasion motor components upon calcium pathway stimulation.

MaxQuant analyses of high mass-accuracy SILAC data reveal that the phosphorylation of at least two sites on Toxoplasma GAP45 (S184/185, T189), two sites on MLC1 (T98, S132) and one on MyoA (S21) increased upon calcium pathway stimulation (H/L ratio normalized - underlined). Intact invasion motor complexes were immunoaffinity purified by anti-GAP45 column chromatography, digested in solution using trypsin, and analysed on an Orbitrap LC-MS/MS mass spectrometer as outlined in Figure 3 and detailed in Materials & Methods. Header rows (bold, italics) list the protein name and ToxoDB entry (ToxoDB release 6.0 ID). Listed below, from left to right: The localized phosphorylation site, phosphopeptide amino acid sequence (best evidence), MaxQuant phos(S/T/Y) localization probability, highest individual Mascot score (best evidence, rank 1), ΔMascot score (best evidence, rank 2), and the average H/L ratio and standard deviation of individual phosphopeptide evidence spectra normalized by protein. (a-j) MS/MS evidence spectra shown in Supplementary Figure S5.