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Combined peptidomic and proteomic analysis of electrically stimulated and manually dissected venom from the South American bullet ant Paraponera clavata Samira R Aili, Axel Touchard, Frédéric Petitclerc, Alain Dejean, Jérôme Orivel, Matthew Paul Padula, Pierre Escoubas, and Graham Michael Nicholson J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00948 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017
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For submission to J Proteome Res
Combined peptidomic and proteomic analysis of electrically stimulated and manually dissected venom from the South American bullet ant Paraponera clavata Samira R. Ailia, Axel Touchardb, Frédéric Petitclercb, Alain Dejeanb,c,, Jérôme Orivelb, Matthew P. Padulad, Pierre Escoubase, Graham M. Nicholsona*
a
Neurotoxin Research Group, School of Life Sciences, University of Technology Sydney, NSW
2007, Australia b
CNRS, UMR Ecologie des Forêts de Guyane (EcoFoG), AgroParisTech, Cirad, INRA, Université
des Antilles, Université de Guyane, Université des Antilles, 97310 Kourou, France c
Ecolab, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France
d
Proteomics Core Facility, Faculty of Science, University of Technology Sydney, NSW 2007,
Australia e
VenomeTech, 473 Route des Dolines — Villa 3, Valbonne 06560, France
*Author for correspondence: Graham Nicholson, Neurotoxin Research Group, School of Life Sciences, University of Technology Sydney, NSW 2007, Australia. Tel: (+61 2) 9514 2230; Fax: (+61 2) 9514 1656 E-mail:
[email protected] (G. Nicholson)
Keywords: Proteome, Peptidome, Ants, Bullet Ant, Venom, MALDI-TOF MS, nanoLC-ESIQTOF MS/MS, 2D-PAGE, Paraponera clavata, dissection, electrical stimulation
ACS Paragon Plus Environment
Journal of Proteome Research
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Abstract Ants have evolved venoms rich in peptides and proteins used for predation, defence and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high performance liquid chromatography then analysed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94) mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with 20 (−10 lgP). Proteins were assigned as homologous when spots were located at the same position (mass and pI) on at least two 2D gels and the protein was identified from the peptide fragment in at least one of the gel spots. Functions were assigned according to the GO annotation within UniProt, where available. Spectra have been provided for proteins matched with a coverage of less than 2% and have been included in the supporting information file (Figure S1-S4). Chemical Reagents All chemicals used were of analytical grade and, unless otherwise stated, were sourced from Sigma Aldrich (NSW, Australia). All buffers were prepared using Milli-Q (18 MΩ/cm2) water.
Results Peptidome Analysis Initially, the venom collected from P. clavata obtained by electrical stimulation and venom gland dissection of colony 1 was subjected to MALDI-TOF MS analysis. Venom obtained by either method was dominated by peptides in the mass range 2600–3200 m/z (Figure 1AaBa). It was also found that no peptides appeared to be present at masses greater than 3200 m/z and that manually dissected venom appeared to contain more peptides than electrically stimulated venom, particularly at masses
