Top Down Proteomics
Reveals Mature Proteoforms Expressed
in Subcellular Fractions of the <i>Echinococcus granulosus</i> Preadult Stage
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Abstract
<i>Echinococcus granulosus</i> 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, <i>E. granulosus</i> 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 <i>E. granulosus</i> 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, <i>in silico</i> 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 <i>E. granulosus</i> 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