Immunoproteomic Identification of Immunogenic Cytoskeleton Proteins of Toxoplasma gondii
Description
In the present study, we analyzed the profile of immunerecognized proteins of the cytoskeleton of T. gondii by 2D PAGE-SDS and sera from patients with acute and chronic toxoplasmosis. The immune-recognized spots were analyzed by mass spectrometry and characterized by bioinformatic approaches. A total of 324 proteins were identified by mass spectrometry, of which sixty-five antigenic proteins were recognized in spots by IgM antibodies and 259 antigenic proteins were recognized by IgG antibodies. About 19 proteins specifically reported as cytoskeletal proteins were identified, as well as numerous hypothetical, secretory proteins with various subcellular functions and distributions. Bioinformatics analyses of the identified antigenic proteins of T. gondii were carried out to determine the most immunogenic as well as the number of epitopes that could be recognized by receptors of B lymphocytes, cytotoxic T lymphocytes (CD8+), and helper T lymphocytes (CD4+), and activate immunoprotection against this pathogen in the quest for a future vaccine against Toxoplasma. This type of experimental approaches will allow us to identify candidate molecules in the design of future vaccines against toxoplasmosis.
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Protein spots recognized by immune sera in WB assays were cut from two-dimensional PAGE-SDS gels, analyzed by mass spectrometry (MS) and performed on a Waters SYNAPT G2-Si high-definition mass spectrometry equipment ( Waters Co., USA). Four exclusion criteria were used to select the identified proteins of interest. The first was based on the analysis of the chromatogram, identifying an exponential relationship with the amount of sample. The second consisted of eliminating non-unique peptides and purifying proteins with confidence values less than 95%. The third consisted of removing peptides with "REVERSE" sequences, and the fourth consisted of proteins that were identified by the presence of at least 3 peptides. *.raw files containing MS and MS/MS spectra were analyzed and quantified with Progenesis QI for Proteomics v4.2 (Waters, Milford, MA, USA) using a combined *.fasta database of the RH strain of T. gondii (UniProt, UP000557509) and the T. gondii GT1 strain (UniProt, UP000005641). The results obtained with Progenesis were exported in *.csv files. For the evaluation of differential protein expression, the base 2 logarithm was used; values of +1 or more indicated increases in expression, and -1 or less indicated decreases.