Then, thiol groups were alkylated with 50 mM iodoacetamide for 1 h at room temperature in the darkness and digested in situ with sequencing grade trypsin (Promega, Madison, WI, USA) as described by Shevchenko et al

Then, thiol groups were alkylated with 50 mM iodoacetamide for 1 h at room temperature in the darkness and digested in situ with sequencing grade trypsin (Promega, Madison, WI, USA) as described by Shevchenko et al. cell-derived trypomastigotes (TCT) of The data obtained reflects the drastic changes in the protein composition as well as the nanomechanical properties of the vesicles produced by both stages of the parasite, differences that could have clear implications in the parasites survival in two radically different biological niches. 2. Results 2.1. Purification and Characterization of EVs To obtain EVs of E and TCT, a procedure including differential centrifugation, coupled to a filtration process through 0.22 m pore filters and ultracentrifugation was employed (Physique S1). After the isolation and purification process, transmission electron microscopy (TEM) analysis revealed the success of the methodology employed in separating the EVs (Physique 1). Regarding nanoparticle tracking analysis (NTA), the mean size of most of the vesicles secreted by E was 259 21 nm and the mode was 1712 11 nm, while the mean size of the vesicles of TCT was 143 24 nm and the Calcipotriol mode was 63 24 nm (Physique 1E,F). These results were similar to measurements obtained by Calcipotriol dynamic light scattering (DLS) in the case of EVs of E (mean size: 183 22 nm); however, in the case of the EVs of TCT, the resulting mean size was smaller (60 17 nm). Thus, it is shown that this EVs secreted by TCT seem to be smaller than the EVs of E but both coincide with the reported size of EVs (Physique 1). Open in a separate window Physique 1 Mouse monoclonal to AURKA Purification of the extracellular vesicles (EVs) of epimastigotes (E) and EVs of tissue-culture cell-derived trypomastigotes (TCT) of (Pan4 strain, DTU I). Scanning electron microscopy (SEM) of E (A) and TCT (B) employed in this study (scale bar: 1 m). (C) Transmission electron microscopy (TEM) of purified EVs of E (scale bar: 1 m). (D) TEM of purified EVs of TCT (scale bar: 500 nm). (E) Nanoparticle tracking analysis (NTA) size distribution of purified EVs of E. (F) NTA size distribution of purified EVs of TCT. Representative figures and graphs of 7 different replicates are shown. 2.2. Proteomic Profile of EVs of E and TCT Western blot analysis using polyclonal anti-antibodies revealed numerous proteins with different expression profiles, indicating the different nature of the protein cargos of EVs of E and EVs of TCT (Physique 2A). To generate an overview of the proteomic differences between the EVs of both stages, seven independent biological replicates (four for EVs of TCT and three for EVs of E) of 40 g purified EVs were subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS). All proteins were searched against UniProt-CL Brener database and proteins present in at least two replicates, with two or more peptides identified, were used for further comparisons. As a result, 528 proteins were identified Calcipotriol in the EVs of TCT and 415 proteins in the EVs of E (Table S1). In either EVs of E or EVs of TCT, the proteins found have different probabilities for appearance in a given sample (2/3 or 3/3 in E and 2/4, 3/4 and 4/4 in TCT). These results indicate that this protein cargos of EVs are either constitutively present or eventually exported through this route (Physique S2). Open in a separate window Physique 2 Qualitative proteomic analysis of the cargos of EVs Calcipotriol of E and EVs of TCT of antibodies. (B) Venn diagram of specific and shared proteins of EVs of E and EVs of TCT. (C) Pie chart representing the percentage of proteins of EVs of E and EVs of TCT that belong to multigene families. (D) Western blot analysis for the confirmation of cruzipain, (1) cruzipain in whole lysates of TCT; (2) cruzipain in EVs of TCT; (3) 0.01). Of those proteins, 274 were present in the EVs of both stages, while.