Quantitative assessment of invasion motor complex components.

Summary of the relative yield of components of immunoaffinity-purified invasion motor complexes. Toxoplasma MyoA, GAP50, GAP45, MLC1, GAP40 and a 15 kDa Calmodulin-like hypothetical protein (TGME49_069440) (ELC1-see text) were the five most abundant Toxoplasma proteins in digests of unlabeled invasion motor complexes, based on the total number of detected spectra. Quantitative analysis of SILAC-labeled motor complex preparations indicates a specific 2–3 fold enrichment in the yield of heavy-labeled invasion motor complex components following Ca2+ stimulation. 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. Listed, from left to right: Protein name, ToxoDB entry (ToxoDB release 6.0 ID), theoretical molecular mass (Mr), sequence coverage (%), the number of unique peptides (sequence/modifications/labels), the total number of spectra (total spectra count), and the average H/L ratio for all detected SILAC pairs estimated using the MaxQuant algorithm [51].