Constructing comprehensive venom proteome reference maps for integrative venomics

Abstract

Objective: Understanding the molecular basis of complex adaptive traits, such as snake venom, demands qualitative and quantitative comparisons of the temporal and spatial patterns of venom variation. Here, we assessed the proof–of-concept that locus-resolved reference venom proteome maps can be achieved through efficient pre-MS venom proteome decomplexation, peptide-centric MS/MS analysis and species-specific database searching. Methods: Venom proteome components were fractionated and quantified by RP-HPLC, SDS-PAGE and 2DE prior to LC-MS/MS matching against a species-specific transcriptomic dataset. Results: Combination of RP-HPLC/SDS-PAGE and 2DE followed by LC-MS/MS showed the existence of ∼178–180 venom protein species generated from ∼48 unique transcripts. Conclusions: Our results underscore that if sufficient pre-MS and MS efforts are applied, comprehensive venom maps can be achieved. And – equally important – dissociating the venom decomplexing steps from the protein identification process represents the key to achieving a quantitative and locus-resolved insight of the venom proteome.

Keywords:

• Crotalus adamanteus
• mass spectrometry
• reverse phase-HPLC
• snake venom proteomics
• two-dimensional electrophoresis
• venomics

Financial & competing interests disclosure

S Eichberg gratefully acknowledges EU Programme ERASMUS for supporting her work at the IBV-CSIC (Valencia, Spain). S Eichberg also acknowledges Prof. Dr.-Ing. Roza Maria Kamp (Technische Fachhochschule Berlin) for her continuous support during this project. Research by JJ Calvete, L Sanz and D Pla was financed by grants BFU2010-17373 from the Ministerio de Ciencia é Innovación and BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid (Spain). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• Developing the full potential of bottom-up venomics requires the integration of pre-MS decomplexation methods (RP-HPLC, SDS-PAGE, 2DE) and tandem mass spectrometric-based protein matching against a comprehensive species-specific transcriptomic database.

• Decomplexation of the venom proteome by RP-HPLC offers the possibility of relative or absolute quantification of venom components.

• 2DE complements the RP-HPLC/SDS-PAGE approach in the characterization of the venom proteome and provides a more accurate view of the macromolecular organization and the existence of proteoforms of venom proteins.

• Comprehensive characterization of the venom proteome may serve as a reference map for integrative venomic studies requiring quantitative locus-specific resolution.

• Percentage compositional data can be transformed into absolute (e g., µg, moles, molecules) or relative (µg/µl) figures not subject to constant-sum constraint, thus allowing the use of standard statistical methods for comparisons of compositional data while retaining the biological meaning of the variables.

• The combination of proteomic and transcriptomic allow the inference a translation rate of toxin-class mRNAs, setting the ground to investigate the basis of the intra- and interspecific differential expression of toxins.

• Top-down mass spectrometry may increasingly gain momentum in proteomic analysis of individual intact protein iso- and proteoforms, and will revolutionize locus-resolved venomics analysis in the coming years.

• Implementation of novel approaches of isotope dilution mass spectrometry to achieve absolute quantification of the individual venom components should become routine in the near future.

• Understanding the natural history and evolutionary pressures and mechanisms that shaped the complexity and diversity of snake venoms is of applied importance for exploring the biotechnological potential of venoms, understanding the molecular mechanisms that underlie venom variability and for the manufacturing of rationally improved therapeutic antivenoms.

Notes