Functional proteomics and bioinformatics to monitor gene expression, identify vaccine targets, and discover novel biochemical pathways in Trypanosoma cruzi
Atwood, James Alexander
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Trypanosoma cruzi (T. cruzi) is a flagellated protozoan parasite endemic to much of Latin America and the causative agent of Chagas' disease. The functional annotation of the T. cruzi genome in vivo is best facilitated by measuring protein expression through proteomic analysis, which not only offers an understanding of stage specific protein expression, but also post-translational modifications. Presented here are proteomic and bioinformatic approaches, all of which center on the high-throughput analysis of T. cruzi gene expression. Herein, we describe a heuristic method for organizing proteins identified at a specified false discovery rate using Mascot matched peptides. We call this method PROVALT. It was evaluated using Mascot identified peptides from a Trypanosoma cruzi epimastigote whole-cell lysate, which were separated by multidimensional liquid chromatography and analyzed by tandem mass spectrometry. PROVALT was shown to be superior to both the single peptide score and cumulative protein score methods. The whole-organism, proteomic analysis of the four life-cycle stages of T. cruzi was performed. Peptides mapping to 2784 proteins in 1168 protein groups from the annotated T. cruzi genome were identified across the four life-cycle stages. Evidence is presented for the expression of protein products from >1000 genes currently annotated as "hypothetical", including members of a gene family annotated as mucin-associated surface proteins (MASPs). Furthermore, this analysis revealed the apparent utilization of distinct energy sources by the individual parasite stages, including histidine for stages present in the insect vectors and fatty acids by intracellular amastigotes. A method for the high-throughput identification of membrane associated N-linked glycoproteins from Trypanosoma cruzi is described. This analysis was based on enrichment of trypomastigote membrane proteins followed by capture of membrane associated N-linked glycoproteins by Concanavalin A affinity chromatography. Through stable isotope labeling of 18the glycan attachment sites with O we unambiguously identified 19 glycopeptides which mapped to 17 glycoproteins, all of which were membrane associated. We also present the first evidence for the expression of 7 putative trypomastigote cell surface glycoproteins including GP-90, DGF-1, and a novel cysteine protease SCP1. We also discuss the implications of two ER localized glycoproteins identified in this analysis, STT3 and GRP94.