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 H₂¹⁸O, 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. Keywords: Trypanosoma cruzi • lectin affinity • stable isotope labeling • glycoproteomics • glycomics • PROVALT • false discovery rate • mucin associated surface protein • dispersed gene family