== Autosampler-based gradient inside a two-dimensional liquid chromatography tandem mass spectrometry system. a Chagas disease vaccine. == 1. Intro == Chagas disease, or American trypanosomiasis, is considered a neglected infectious disease with an estimated 11 million instances in Latin America, and about 50,000 deaths yearly.13Chagas disease has become a public PD158780 health concern in the U.S. and Europe due to the large number of asymptomatic infected people migrating from endemic areas.4,5In addition, autochthonous cases have been reported in the U.S.4,6The etiologic agent of Chagas disease is the protozoan parasiteTrypanosoma cruzi, which has four life-cycle stages or forms, namely epimastigotes, metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes, in two unique hosts, i.e., triatomine insect and mammal.1,3In the natural cycle, infective bloodstream trypomastigote forms are ingested from the triatomine insect vector (popularly known as thekissing bug) during a blood meal. In the bugs midgut, trypomastigotes differentiate into noninfective epimastigote forms, which reproduce by binary fission. In the posterior region of the gut (hindgut), upon nutritional stress epimastigotes transform into infective metacyclic trypomastigote forms, which are then released with the excreta during a blood meal. These parasites then penetrate into the sponsor bloodstream through the bite wound or revealed mucosa and infect a variety of nucleated cells. Once inside the cell, the metacyclic trypomastigote immediately escapes the parasitophorous vacuole and transforms into proliferative amastigote form, which replicates by asexual binary division. After several division cycles, amastigotes differentiate into trypomastigotes, which are then released to the extracellular milieu, eventually reaching the bloodstream to infect fresh cells or become ingested from the kissing bug, completing the natural life cycle.1,3 Current therapy against Chagas disease is based only in two medicines: benznidazole and nifurtimox. Both these compounds have partial effectiveness in the chronic phase of the disease and PD158780 severe side effects.7,8Moreover, thus far there is no available vaccine for the treatment and/or prevention of human being Chagas disease.911In recent years, several studies have focused in the host immune response againstT. cruzi.1214Although some encouraging progress have been made, the difficulty of using a more rational approach to develop a vaccine againstT. cruziis the restricted information PD158780 about proteins expressed around the cell surface of different parasite stages, strains, and phylogenetic lineages.15 T. cruziis covered Rabbit Polyclonal to SIN3B by a thick layer of glycosylphosphatidylinositol (GPI)-anchored molecules, which are encoded by multigene families, such as mucins (TcMUC), mucin-associated surface proteins (MASP),trans-sialidase (TS)/gp85, and gp63 glycoproteins, each one of them with hundreds of members.1619However, the lack of positive correlation between the levels of transcripts and proteins makes difficult the use of PD158780 approaches such as microarray and cDNA library to map the expressed sequences.20Thus, the use of proteomic approaches is an interesting and reliable alternative to analyze gene expression inT. cruzi. For instance, a large-scale proteomic study ofT. cruziBrazil strain identified 1,168 non-redundant proteins (2,784 individual proteins) combing the data from all four stages PD158780 of the parasite.21 Multidimensional liquid chromatography (MDLC) coupled to tandem mass spectrometry (MS/MS) for proteomic analysis was first introduced by John Yates group and became one of the most popular approaches to analyze large-scale proteomic samples.22In this approach, proteins from complex mixture samples are chemically or enzymatically digested, resulting in thousands of peptides, which are then analyzed by.