RESUMO
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.
Assuntos
Proteômica , Trypanosoma cruzi/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Sequência de Aminoácidos , Animais , Western Blotting , Fracionamento Celular , Cromatografia de Afinidade , Cromatografia Líquida , Biologia Computacional , Lectinas , Espectrometria de Massas , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional/genéticaRESUMO
We describe an algorithm which modifies a protein database such that during a database search deamidation is limited to asparagines strictly contained within the N-glycosylation consensus sequence. The modified database was evaluated using a dataset created from the shotgun proteomic analysis of N-linked glycopeptides from human blood serum. We demonstrate that the application of the modified database eliminates incorrect glycopeptide assignments, reduces the peptide false-discovery rate, and eliminates the need for manual validation of glycopeptide identifications.
