title: Quantitative proteomic and phosphoproteomic analysis of Trypanosoma cruzi amastigogenesis.
abstract: Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets.
authors.affiliation: From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910-900 Brazil; §Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Rayner M L
authors.initials: RM
authors.lastname: Queiroz
authors.affiliation: From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910-900 Brazil;
authors.forename: Sébastien
authors.initials: S
authors.lastname: Charneau
authors.affiliation: From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910-900 Brazil;
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Mandacaru
authors.affiliation: §Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Veit
authors.initials: V
authors.lastname: Schwämmle
authors.affiliation: From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910-900 Brazil;
authors.forename: Beatriz D
authors.initials: BD
authors.lastname: Lima
authors.affiliation: §Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.affiliation: From the ‡Department of Cell Biology, Institute of Biology, University of Brasilia, Brasília, 70910-900 Brazil; ricart@unb.br.
authors.forename: Carlos A O
authors.initials: CA
authors.lastname: Ricart
mesh_terms: D003470:Culture Media / Q000494:pharmacology; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D008018:Life Cycle Stages / Q000187:drug effects / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D058977:Molecular Sequence Annotation; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D003470:Culture Media; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D015800:Protozoan Proteins; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases