Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.
Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.
Atwood
James A
JA
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602-4712, USA.
Minning
Todd
T
Ludolf
Fernanda
F
Nuccio
Arthur
A
Weatherly
Daniel B
DB
Alvarez-Manilla
Gerardo
G
Tarleton
Rick
R
Orlando
Ron
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D019295:Computational Biology; D037102:Lectins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000235:genetics; D040901:Proteomics; D014349:Trypanosoma cruzi / Q000235:genetics*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
D037102:Lectins; D014643:Variant Surface Glycoproteins, Trypanosoma