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. 2015 Nov 6;14(11):4805-14.
doi: 10.1021/acs.jproteome.5b00642. Epub 2015 Oct 28.

Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage

Karina R Lorenzatto  1 , Kyunggon Kim  2 , Ioanna Ntai  2 , Gabriela P Paludo  1 , Jeferson Camargo de Lima  1 , Paul M Thomas  2 , Neil L Kelleher  2 , Henrique B Ferreira  1
Affiliations

Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage

Karina R Lorenzatto et al. J Proteome Res. .

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.

Keywords: hydatid disease; low mass proteome; post-translational modifications; subcellular fractions; top down proteomics.

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Figures

Figure 1
Figure 1
Representative data from LC–MS/MS analysis of a single GELFrEE fraction. (A) GELFrEE fractionation of the nuclear sample shown as an example. (B) LC total ion chromatogram from GELFrEE fraction 2. (C) two representative spectra from intact proteins.
Figure 2
Figure 2
Overlapping between nuclear and cytosolic protein IDs and proteoforms identified by the top down approach. The comparison of proteoforms points out that, although some IDs are common, they exhibit modifications that make them different proteoforms, explaining the decrease in the overlap between nuclear and cytosolic fraction.
Figure 3
Figure 3
Comparison between proteins identified using top down and bottom up approaches. (A) Venn diagram showing a partial overlap between the approaches. (B) molecular mass distribution of proteins identified in both approaches.
Figure 4
Figure 4
Overview of the CTMs and sequence motifs detected in the identified proteoforms. (A) Venn diagram showing the distribution of NME and NTA events. The overlap indicate proteoforms modified by NME + NTA. (B) Sequence motifs for the first 10 residues of each proteoform identified displaying NME only, NME + NTA (NatA putative substrates), and NTA only (NatC-F putative substrates).
Figure 5
Figure 5
Detection of two proteoforms from histone H2A. These monoacetylated and diacetylated proteoforms were only detected using our modified database including the acetylation prediction information.
Figure 6
Figure 6
Overview of the low mass proteome from E. granulosus protoscoleces subcellular fractions. (A) Distribution of nuclear and cytosolic proteins identified using both top down and bottom up MS. (B) gene ontology (GO) classification of proteins identified in E. granulosus subcellular fractions. The results of BlastP searches against the NCBInr database were used for GO mapping and annotation. Top 10 most abundant GO terms for cellular component are shown.
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