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Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and stra...

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Autores principales: Amorim, Fernanda Gobbi, Redureau, Damien, Crasset, Thomas, Freuville, Lou, Baiwir, Dominique, Mazzucchelli, Gabriel, Menzies, Stefanie K., Casewell, Nicholas R., Quinton, Loïc
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10304959/
https://www.ncbi.nlm.nih.gov/pubmed/37368658
http://dx.doi.org/10.3390/toxins15060357
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author Amorim, Fernanda Gobbi
Redureau, Damien
Crasset, Thomas
Freuville, Lou
Baiwir, Dominique
Mazzucchelli, Gabriel
Menzies, Stefanie K.
Casewell, Nicholas R.
Quinton, Loïc
author_facet Amorim, Fernanda Gobbi
Redureau, Damien
Crasset, Thomas
Freuville, Lou
Baiwir, Dominique
Mazzucchelli, Gabriel
Menzies, Stefanie K.
Casewell, Nicholas R.
Quinton, Loïc
author_sort Amorim, Fernanda Gobbi
collection PubMed
description To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.
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spelling pubmed-103049592023-06-29 Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal Amorim, Fernanda Gobbi Redureau, Damien Crasset, Thomas Freuville, Lou Baiwir, Dominique Mazzucchelli, Gabriel Menzies, Stefanie K. Casewell, Nicholas R. Quinton, Loïc Toxins (Basel) Article To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition. MDPI 2023-05-25 /pmc/articles/PMC10304959/ /pubmed/37368658 http://dx.doi.org/10.3390/toxins15060357 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Amorim, Fernanda Gobbi
Redureau, Damien
Crasset, Thomas
Freuville, Lou
Baiwir, Dominique
Mazzucchelli, Gabriel
Menzies, Stefanie K.
Casewell, Nicholas R.
Quinton, Loïc
Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title_full Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title_fullStr Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title_full_unstemmed Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title_short Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal
title_sort next-generation sequencing for venomics: application of multi-enzymatic limited digestion for inventorying the snake venom arsenal
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10304959/
https://www.ncbi.nlm.nih.gov/pubmed/37368658
http://dx.doi.org/10.3390/toxins15060357
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