title: The identification and characterization of epitopes in the 30-34 kDa Trypanosoma cruzi proteins recognized by antibodies in the serum samples of chagasic patients.
abstract: Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: giovaniverissimo@gmail.br.
authors.forename: Giovani Carlo
authors.initials: GC
authors.lastname: Verissimo da Costa
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Leticia Miranda Santos
authors.initials: LM
authors.lastname: Lery
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Manuela Leal
authors.initials: ML
authors.lastname: da Silva
authors.affiliation: Biological Mass Spectrometry Laboratory, ERAT Branch, Centers of Disease Control and Prevention, Atlanta, GA, USA; Division of Laboratory Sciences, National Center of Enviromental Health, Centers of Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Hércules
authors.initials: H
authors.lastname: Moura
authors.affiliation: Departamento de Patologia, Faculdade de Medicina, Universidade Federal Fluminense, Niterói, Rio de Janeiro, Brazil.
authors.forename: Regina Helena Saramago
authors.initials: RH
authors.lastname: Peralta
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Wanda Maria Almeida
authors.initials: WM
authors.lastname: von Krüger
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Paulo Mascarello
authors.initials: PM
authors.lastname: Bisch
authors.affiliation: Biological Mass Spectrometry Laboratory, ERAT Branch, Centers of Disease Control and Prevention, Atlanta, GA, USA; Division of Laboratory Sciences, National Center of Enviromental Health, Centers of Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Peralta
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D014355:Chagas Disease / Q000097:blood* / Q000469:parasitology*; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry*; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D020543:Proteome; D040901:Proteomics / Q000379:methods; D014349:Trypanosoma cruzi / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D018985:Epitopes, B-Lymphocyte; D007074:Immunoglobulin G; D010455:Peptides; D020543:Proteome