title: Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.
abstract: 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.
authors.affiliation: Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul , Avenida Bento Gonçalves, 9500 Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Karina R
authors.initials: KR
authors.lastname: Lorenzatto
authors.affiliation: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Kyunggon
authors.initials: K
authors.lastname: Kim
authors.affiliation: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.affiliation: Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul , Avenida Bento Gonçalves, 9500 Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Gabriela P
authors.initials: GP
authors.lastname: Paludo
authors.affiliation: Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul , Avenida Bento Gonçalves, 9500 Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Jeferson
authors.initials: J
authors.lastname: Camargo de Lima
authors.affiliation: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.affiliation: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University , 2145 North Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul , Avenida Bento Gonçalves, 9500 Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Henrique B
authors.initials: HB
authors.lastname: Ferreira
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry / Q000469:parasitology; D002853:Chromatography, Liquid; D003600:Cytosol / Q000737:chemistry / Q000469:parasitology; D004443:Echinococcosis / Q000469:parasitology / Q000473:pathology; D048209:Echinococcus granulosus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004847:Epithelial Cells / Q000737:chemistry / Q000469:parasitology; D015801:Helminth Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008018:Life Cycle Stages / Q000235:genetics; D008168:Lung / Q000737:chemistry / Q000469:parasitology; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D015801:Helminth Proteins; D006657:Histones; D020543:Proteome; D008715:Methionine
keywords: hydatid disease; low mass proteome; post-translational modifications; subcellular fractions; top down proteomics