Proteins were separated on 24 × 18-cm Tricine/SDS-PAGE (12% acrylamide) [77].
After migration, the gels were fixed, and the proteins were visualized by coomassie brilliant blue R-250. Images of the gels were taken with a high-resolution scanner (Amersham Biosciences). BN-PAGE Proteins were concentrated and directly loaded on native PAGE gradients 6-15% acrylamide for the first dimension and on a 12% Tricine-SDS-PAGE for the second dimension, as described in Peltier et al., 2004 [78]. Proteins were visualized by coomassie blue staining. Protein identification by mass spectrometry Stained protein spots were manually excised, washed, digested with trypsin, and extracted using formic acid. Protein digests were analyzed using either a hybrid triple-quadrupole linear ion trap mass spectrometer (Q-TRAP 4000; Applied Selleck LY2874455 Biosystems), coupled to a nano-chromatography system (Dionex), or an ion trap mass spectrometer (Esquire HCT; Bruker), interfaced with an HPLC (high-performance liquid chromatography) chip system (Agilent). MS/MS data were searched against NCBI (National Center for Biotechnology Information) and Trypanosoma
brucei databases using Mascot software. Raw data were analyzed using Data Analysis software (Bruker) to generate a peak list for searching a Trypanosoma database extracted from the Sanger Geneticin supplier Institute. The Mascot (v2.2) search engine was used with the following parameters: one missed cleavage allowed for trypsin, PDK4 carboxymethylation of cyst as fixed modification, methionine oxidation as variable AG-881 price modification, and a 0.6-Da tolerance range for mass accuracy in MS/MS. At least one matching sequence tags of high quality was needed for positive identification of proteins. Potential false positive identifications have been addressed as described in Elias et al., 2005 [79] using identical search parameters against a database in which the sequences have been reversed. We set a false discovery rate (FDR) of 1%. When the Mascot peptide score was below (and even above) the Mascot peptide score indicated for a FDR of 1%, a systematic manual validation was done with stringent parameters (at least 6 y or b ions, at least
4 consecutive ions, and peptidic sequence formed of more than 7 amino acids). The proteins were classified according to MapMan http://mapman.gabipd.org. Raw data will be made available upon request for research purposes. Additional data on identified proteins are supplied in additional file 8 (Table S8). Preparation of vesicles by ultracentrifugation and sucrose gradient Secretion buffer and infected rat serum after parasite depletion were filtered (0.2 μm). Membranes were isolated from secretion buffer and serum of Trypanosoma-infected rats by a 140,000 g ultracentrifugation for 30 min at 4°C. Pellets were resuspended in 20 mM Tris/Hcl buffer pH 7.8 and layered on top of a step sucrose gradient (20-30-40-60% sucrose [Sigma-Aldrich]).