RESUMEN

The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

Asunto(s)

Bothrops , Venenos de Crotálidos , Animales , Bothrops/metabolismo , Proteómica/métodos , Brasil , Venenos de Crotálidos/química , Espectrometría de Masas , Proteoma/análisis

RESUMEN

Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

Asunto(s)

Echinococcus granulosus/metabolismo , Proteínas del Helminto/aislamiento & purificación , Histonas/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Proteoma/aislamiento & purificación , Proteómica/métodos , Acetilación , Secuencia de Aminoácidos , Animales , Bovinos , Núcleo Celular/química , Núcleo Celular/parasitología , Cromatografía Liquida , Citosol/química , Citosol/parasitología , Equinococosis/parasitología , Equinococosis/patología , Echinococcus granulosus/genética , Echinococcus granulosus/crecimiento & desarrollo , Células Epiteliales/química , Células Epiteliales/parasitología , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Histonas/genética , Histonas/metabolismo , Estadios del Ciclo de Vida/genética , Pulmón/química , Pulmón/parasitología , Metionina/química , Metionina/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Proteómica/instrumentación , Espectrometría de Masas en Tándem