Glycoproteomics of<i>Trypanosoma cruzi</i>Trypomastigotes Using Subcellular Fractionation, Lectin Affinity, and Stable Isotope Labeling

Atwood, James; Minning, Todd; Ludolf, Fernanda; Nuccio, Arthur; Weatherly, D. Brent; Álvarez-Manilla, Gerardo; Tarleton, Rick L.; Orlando, Ron

doi:10.1021/pr060364b

Cited by 74 publications

(85 citation statements)

References 43 publications

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“…6% of the total parasite diploid genome (9). Nevertheless, only 14 MASP proteins have been identified to date by proteomic identification made via a single peptide match (5,7,19), which suggests that this gene family either does not express itself as abundantly as the TS proteins or that they undergo posttranscriptional modifications, as do the mucins, which makes their detection difficult by shotgun proteomics. Subsequent sequencing and proteomic approaches to MASP52 resulted in seven matches, which corresponded to a putative MASP, with the EMBL accession number XP_820015.1 (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The specificity of this family for T. cruzi, the diversity of the masp genes, and their expression by the infective stages of the parasite, together with the existence of hypervariable regions, all lead us to believe that some of the proteins encoded by these genes may well be involved in the invasion process and establishment of the parasite within the host cell (9,18). Just as the TS, gp82, Tc-1, and POPTc80 proteins have been identified through the use of antibodies as being essential to cell invasion (5,8,10,36,45), the results of our assays using specific antibodies against the catalytic region (Fig. 7) by reducing significantly the percentages of parasitization.…”

Section: Discussionmentioning

confidence: 99%

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Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

et al. 2011

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We describe the characterization, purification, expression, and location of a 52-kDa protein secreted during interaction between the metacyclic form of Trypanosoma cruzi and its target host cell. The protein, which we have named MASP52, belongs to the family of mucin-associated surface proteins (MASPs). The highest levels of expression of both the protein and mRNA occur during the metacyclic and bloodstream trypomastigote stages, the forms that infect the vertebrate host cells. The protein is located in the plasma membrane and in the flagellar pockets of the epimastigote, metacyclic, and trypomastigote forms and is secreted into the medium at the point of contact between the parasite and the cell membrane, as well as into the host-cell cytosol during the amastigote stage. IgG antibodies specific against a synthetic peptide corresponding to the catalytic zone of MASP52 significantly reduce the parasite's capacity to infect the host cells. Furthermore, when the protein is adsorbed onto inert particles of bentonite and incubated with a nonphagocytic cell culture, the particles are able to induce endocytosis in the cells, which seems to demonstrate that MASP52 plays a role in a process whereby the trypomastigote forms of the parasite invade the host cell.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

et al. 2011

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“…Isotope labeling oligosaccharides in the mass spectrometric analyses is also quite useful for the identiˆcation of glycosylation sites (69)(70)(71) and quantitative analysis of oligosaccharide mixtures (72,73). It is, however, not feasible to determine the oligosaccharide structure unambiguously solely on the MS data because there exist a variety of isomeric structures with diŠerent composition, glycosidic linkage and branching.…”

Section: E Mass Spectrometric Analysis Of Isotope-labeled Glycansmentioning

confidence: 99%

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Yamaguchi¹

2008

TIGG

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“…One approach uses lectin affinity chromatography (23,24), while others take advantage of the specific reactivity of oxidized glycosyl groups with hydrazine-activated solid supports (25). As with biotinylation, rigorous washing is essential because a number of secreted proteins are also glycosylated.…”

Section: Enrichment Strategiesmentioning

confidence: 99%

Thematic Review Series: Proteomics. Proteomic analysis of lipid-protein complexes

Vaisar

2009

Journal of Lipid Research

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There is intense interest in comprehensive proteomic approaches for analyzing integral membrane proteins and lipoproteins. Key features of mass spectrometric analysis center on enriching biological material for proteins of interest, efficiently digesting them, extracting the resulting peptides, and using fractionation methods to comprehensively sample proteins or peptides by tandem mass spectrometry. However, lipid-associated proteins are generally rich in hydrophobic domains and are often low in abundance. These features, together with the associated lipid, make their mass spectrometric analysis technically challenging. In this article, we review analytical strategies for successful proteomic analysis of lipid-associated proteins. Now that the genomes of humans and many model organisms have been fully sequenced, scientists must determine the molecular and cellular functions of the hundreds of thousands of proteins encoded by those genomes. They would also like to explain how the proteins cooperate or otherwise interact in complex physiological systems and how inappropriate interactions trigger human disease. These important biological problems are the central focus of the rapidly emerging field of proteomics, the study of protein expression, structure, and function.Mass spectrometry (MS) is a powerful tool for proteomics because it can identify and quantify hundreds or even thousands of proteins in complex biological samples. To study peptides and proteins with MS, it is necessary to vaporize these normally involatile compounds. Therefore, a key element in the development of MS-based proteomics, recognized by the 2002 Nobel Prize in Chemistry, was the discovery of methods for introducing peptides and proteins into the gas phase (1, 2).The two most common ionization techniques are ESI and MALDI. ESI is typically applied to analytes in the liquid phase and is often used in concert with separation techniques, such as reverse-phase chromatography. Therefore, it is well suited to the analysis of complex mixtures. MALDI, which uses laser pulses to vaporize analytes embedded in a crystalline matrix, is generally applied to relatively simple mixtures of peptides or proteins.Although protein identification based on tandem mass spectrometric (MS/MS) analysis of intact proteins is an ideal approach, this so-called "top-down" proteomic method (3) is still being developed and is not amenable to largescale investigations. Most proteomic studies take a "bottomup" approach (4) by first digesting proteins with a protease (typically trypsin). The resulting small peptides are much more amenable to separation [typically by liquid chromatography (LC)] and MS/MS analysis.Two general approaches have evolved for protein analysis: gel-based proteomics and gel-free proteomics. The first methodology is widely used by biological investigators and involves various forms of electrophoretic gel-based separations, primarily one-dimensional (e.g., SDS-PAGE) or two-dimensional electrophoresis (2DE). Separated protein spots (or proteoly...

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Glycoproteomics ofTrypanosoma cruziTrypomastigotes Using Subcellular Fractionation, Lectin Affinity, and Stable Isotope Labeling

Cited by 74 publications

References 43 publications

Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

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Thematic Review Series: Proteomics. Proteomic analysis of lipid-protein complexes

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