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Reto Stocklin - One of the best experts on this subject based on the ideXlab platform.
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uncovering intense protein diversification in a cone Snail Venom gland using an integrative Venomics approach
Journal of Proteome Research, 2015Co-Authors: Daniel Biass, Philippe Favreau, Aude Violette, Nicolas Hulo, Frédérique Lisacek, Reto StocklinAbstract:Marine cone Snail Venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the Venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative Venomics approach, based on new proteomic results from the Venom gland and data previously obtained from the transcriptome and the injectable Venom. We describe here the complete peptide content of the dissected Venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the Venom gland. In addition, by integrating data from three different Venom stages, transcriptome, dissected, and injectable Venoms, from a single species, we obtain a global overview of the Venom processing that occurs from the Venom gland tissue to the Venom delivery step. In the light of the successive steps in this Venom production system, we demonstrate that each Venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.
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peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time of flight mass spectrometry application to the conus consors cone Snail Venom
Journal of Chromatography A, 2012Co-Authors: Philippe J Eugster, Daniel Biass, Reto Stocklin, Philippe Favreau, Davy Guillarme, Jeanluc WolfenderAbstract:The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the Venom of the predatory marine Snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors Venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors Venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors Venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.
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conus consors Snail Venom proteomics proposes functions pathways and novel families involved in its Venomic system
Journal of Proteome Research, 2012Co-Authors: Adrijana Leonardi, Daniel Biass, Dusan Kordis, Reto Stocklin, Philippe Favreau, Igor KrižajAbstract:For some decades, cone Snail Venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in Venoms of mollusks. In order to shed more light on cone Snail Venom HMM components, the proteins of dissected and injected Venom of a fish-hunting cone Snail, Conus consors, were extensively assessed. HMM Venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors Venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the Venom duct and assigned a biological function, thus pointing to their potential role in Venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups ...
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Large-scale discovery of conopeptides and conoproteins in the injectable Venom of a fish-hunting cone Snail using a combined proteomic and transcriptomic approach
Journal of Proteomics, 2012Co-Authors: Aude Violette, Sebastien Dutertre, Daniel Biass, Reto Stocklin, David Piquemal, Dominique Koua, Fabien Pierrat, Philippe FavreauAbstract:Predatory marine Snails of the genus Conus use Venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable Venom from Conus consors, an indo-pacific fish-hunting cone Snail. By matching MS/MS data against an extensive set of Venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the Venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable Venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone Snail Venom to date, and delineate the major protein families present in the delivered Venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.
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high resolution picture of a Venom gland transcriptome case study with the marine Snail conus consors
Toxicon, 2012Co-Authors: Yves Terrat, Sebastien Dutertre, Daniel Biass, Reto Stocklin, Philippe Favreau, Maido Remm, David Piquemal, Frederic DucancelAbstract:Although cone Snail Venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in Venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole Venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using Venom ducts of Conus consors, an Indo-Pacific living piscivorous cone Snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in Venom function could be identified for the first time in cone Snail Venom duct. This work provides one of the first large-scale EST project for a cone Snail Venom duct using next-generation sequencing, allowing a detailed overview of the Venom duct transcripts. This leads to an expanded definition of the overall cone Snail Venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the Venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution.
Sebastien Dutertre - One of the best experts on this subject based on the ideXlab platform.
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Revising the Role of Defense and Predation in Cone Snail Venom Evolution
Evolution of Venomous Animals and Their Toxins, 2017Co-Authors: Jutty Rajan Prashanth, Sebastien Dutertre, Richard J. LewisAbstract:Venoms are widely employed by numerous animals across disparate lineages for predation and defense. Among them, the deadly carnivorous cone Snails are reputed for the potency of their Venoms comprising small neurotoxic peptides known as conotoxins. Though a majority of cone Snails prey on worms, some species prey on fish and other mollusks despite being slow movers. This remarkable prey diversification contributes to their evolutionary success. The origins of these dietary shifts have historically been explained based on the synergistic pharmacology of toxin classes. However, the recent discovery that some mollusk- and fish-hunting Snails inject distinct defensive and predatory Venoms has led to an alternative hypothesis where defense plays a pivotal role in the evolution of conotoxins and cone Snails. This chapter provides an overview of cone Snails and highlights recent advances in our understanding of conotoxin evolution.
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Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom
Molecular and Cellular Proteomics, 2013Co-Authors: Sebastien Dutertre, Ai-hua Jin, Quentin Kaas, Alun Jones, Paul Alewood, Richard LewisAbstract:Cone Snails produce highly complex Venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 Venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the Venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected Venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of Venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone Snail.
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Large-scale discovery of conopeptides and conoproteins in the injectable Venom of a fish-hunting cone Snail using a combined proteomic and transcriptomic approach
Journal of Proteomics, 2012Co-Authors: Aude Violette, Sebastien Dutertre, Daniel Biass, Reto Stocklin, David Piquemal, Dominique Koua, Fabien Pierrat, Philippe FavreauAbstract:Predatory marine Snails of the genus Conus use Venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable Venom from Conus consors, an indo-pacific fish-hunting cone Snail. By matching MS/MS data against an extensive set of Venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the Venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable Venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone Snail Venom to date, and delineate the major protein families present in the delivered Venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.
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Conus Venom peptide pharmacology.
Pharmacological reviews, 2012Co-Authors: Richard J. Lewis, Sebastien Dutertre, Macdonald J. ChristieAbstract:Conopeptides are a diverse group of recently evolved Venom peptides used for prey capture and/or defense. Each species of cone Snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone Snail Venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.
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high resolution picture of a Venom gland transcriptome case study with the marine Snail conus consors
Toxicon, 2012Co-Authors: Yves Terrat, Sebastien Dutertre, Daniel Biass, Reto Stocklin, Philippe Favreau, Maido Remm, David Piquemal, Frederic DucancelAbstract:Although cone Snail Venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in Venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole Venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using Venom ducts of Conus consors, an Indo-Pacific living piscivorous cone Snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in Venom function could be identified for the first time in cone Snail Venom duct. This work provides one of the first large-scale EST project for a cone Snail Venom duct using next-generation sequencing, allowing a detailed overview of the Venom duct transcripts. This leads to an expanded definition of the overall cone Snail Venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the Venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution.
P Balaram - One of the best experts on this subject based on the ideXlab platform.
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cone Snail analogs of the pituitary hormones oxytocin vasopressin and their carrier protein neurophysin proteomic and transcriptomic identification of conopressins and conophysins
Biochimica et Biophysica Acta, 2020Co-Authors: Sanjeev Kumar, M Vijayasarathy, M A Venkatesha, P Sunita, P BalaramAbstract:Transcriptomic analysis of cone Snail Venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the Venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.
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cone Snail prolyl 4 hydroxylase α subunit sequences derived from transcriptomic data and mass spectrometric analysis of variable proline hydroxylation in c amadis Venom
Journal of Proteomics, 2019Co-Authors: M Vijayasarathy, P BalaramAbstract:Putative prolyl-4-hydroxylase (P4H) α-subunit sequences have been extracted by mining transcriptomic data obtained from seven cone Snail species C. amadis, C. monile, C. araneosus, C. miles, C. litteratus, C. frigidus, and C. ebraeus. Sequences ranging from 518 to 559 residues have been compared with representative animal P4H sequences. The α-subunit consists of an N-terminus double domain, involved in dimerization and substrate binding, while the C-terminus contains the catalytic domain. Definitive functional annotation of the cone Snail sequences has been achieved by an analysis of conserved residues responsible for catalytic function, specific conformational features, and subunit interactions, using two independent structures of the double domain, and the catalytic domain, previously reported in the literature. The variability of proline hydroxylation in conotoxins is illustrated by a mass spectrometric analysis of C. amadis Venom. Site specific hydroxylation and the presence of peptides with multiple proline residues, resistant to modification, suggests that sequence and conformational effects may determine the substrate specificity of the Conus prolyl-4-hydroxylases. SIGNIFICANCE: Proline hydroxylation is a widely observed post translational modification, with collagen being the pre-eminent example. Hydroxylation of proline is also widely observed in conotoxins, which are a major component of marine cone Snail Venom. This paper describes newly identified prolyl-4-hydroxylase sequences, using transcriptome data from seven Conus species. The predicted functional annotation of prolyl-4-hydroxylase sequences was carried out using two available crystal structures of independent domains. The mass spectrometric characterisation of proline/hydroxyproline containing peptides in C. amadis Venom confirms sequence specific hydroxylation in Conus Venom as shown previously by others.
Daniel Biass - One of the best experts on this subject based on the ideXlab platform.
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uncovering intense protein diversification in a cone Snail Venom gland using an integrative Venomics approach
Journal of Proteome Research, 2015Co-Authors: Daniel Biass, Philippe Favreau, Aude Violette, Nicolas Hulo, Frédérique Lisacek, Reto StocklinAbstract:Marine cone Snail Venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the Venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative Venomics approach, based on new proteomic results from the Venom gland and data previously obtained from the transcriptome and the injectable Venom. We describe here the complete peptide content of the dissected Venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the Venom gland. In addition, by integrating data from three different Venom stages, transcriptome, dissected, and injectable Venoms, from a single species, we obtain a global overview of the Venom processing that occurs from the Venom gland tissue to the Venom delivery step. In the light of the successive steps in this Venom production system, we demonstrate that each Venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.
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Uncovering Intense Protein Diversification in a Cone Snail Venom Gland Using an Integrative Venomics Approach
2015Co-Authors: Daniel Biass, Philippe Favreau, Aude Violette, Nicolas Hulo, Frédérique Lisacek, Reto StöcklinAbstract:Marine cone Snail Venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the Venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative Venomics approach, based on new proteomic results from the Venom gland and data previously obtained from the transcriptome and the injectable Venom. We describe here the complete peptide content of the dissected Venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the Venom gland. In addition, by integrating data from three different Venom stages, transcriptome, dissected, and injectable Venoms, from a single species, we obtain a global overview of the Venom processing that occurs from the Venom gland tissue to the Venom delivery step. In the light of the successive steps in this Venom production system, we demonstrate that each Venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal
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peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time of flight mass spectrometry application to the conus consors cone Snail Venom
Journal of Chromatography A, 2012Co-Authors: Philippe J Eugster, Daniel Biass, Reto Stocklin, Philippe Favreau, Davy Guillarme, Jeanluc WolfenderAbstract:The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the Venom of the predatory marine Snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors Venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors Venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors Venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.
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conus consors Snail Venom proteomics proposes functions pathways and novel families involved in its Venomic system
Journal of Proteome Research, 2012Co-Authors: Adrijana Leonardi, Daniel Biass, Dusan Kordis, Reto Stocklin, Philippe Favreau, Igor KrižajAbstract:For some decades, cone Snail Venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in Venoms of mollusks. In order to shed more light on cone Snail Venom HMM components, the proteins of dissected and injected Venom of a fish-hunting cone Snail, Conus consors, were extensively assessed. HMM Venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors Venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the Venom duct and assigned a biological function, thus pointing to their potential role in Venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups ...
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Large-scale discovery of conopeptides and conoproteins in the injectable Venom of a fish-hunting cone Snail using a combined proteomic and transcriptomic approach
Journal of Proteomics, 2012Co-Authors: Aude Violette, Sebastien Dutertre, Daniel Biass, Reto Stocklin, David Piquemal, Dominique Koua, Fabien Pierrat, Philippe FavreauAbstract:Predatory marine Snails of the genus Conus use Venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable Venom from Conus consors, an indo-pacific fish-hunting cone Snail. By matching MS/MS data against an extensive set of Venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the Venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable Venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone Snail Venom to date, and delineate the major protein families present in the delivered Venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.
Philippe Favreau - One of the best experts on this subject based on the ideXlab platform.
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uncovering intense protein diversification in a cone Snail Venom gland using an integrative Venomics approach
Journal of Proteome Research, 2015Co-Authors: Daniel Biass, Philippe Favreau, Aude Violette, Nicolas Hulo, Frédérique Lisacek, Reto StocklinAbstract:Marine cone Snail Venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the Venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative Venomics approach, based on new proteomic results from the Venom gland and data previously obtained from the transcriptome and the injectable Venom. We describe here the complete peptide content of the dissected Venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the Venom gland. In addition, by integrating data from three different Venom stages, transcriptome, dissected, and injectable Venoms, from a single species, we obtain a global overview of the Venom processing that occurs from the Venom gland tissue to the Venom delivery step. In the light of the successive steps in this Venom production system, we demonstrate that each Venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.
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Uncovering Intense Protein Diversification in a Cone Snail Venom Gland Using an Integrative Venomics Approach
2015Co-Authors: Daniel Biass, Philippe Favreau, Aude Violette, Nicolas Hulo, Frédérique Lisacek, Reto StöcklinAbstract:Marine cone Snail Venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the Venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative Venomics approach, based on new proteomic results from the Venom gland and data previously obtained from the transcriptome and the injectable Venom. We describe here the complete peptide content of the dissected Venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the Venom gland. In addition, by integrating data from three different Venom stages, transcriptome, dissected, and injectable Venoms, from a single species, we obtain a global overview of the Venom processing that occurs from the Venom gland tissue to the Venom delivery step. In the light of the successive steps in this Venom production system, we demonstrate that each Venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal
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peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time of flight mass spectrometry application to the conus consors cone Snail Venom
Journal of Chromatography A, 2012Co-Authors: Philippe J Eugster, Daniel Biass, Reto Stocklin, Philippe Favreau, Davy Guillarme, Jeanluc WolfenderAbstract:The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the Venom of the predatory marine Snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors Venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors Venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors Venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.
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conus consors Snail Venom proteomics proposes functions pathways and novel families involved in its Venomic system
Journal of Proteome Research, 2012Co-Authors: Adrijana Leonardi, Daniel Biass, Dusan Kordis, Reto Stocklin, Philippe Favreau, Igor KrižajAbstract:For some decades, cone Snail Venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in Venoms of mollusks. In order to shed more light on cone Snail Venom HMM components, the proteins of dissected and injected Venom of a fish-hunting cone Snail, Conus consors, were extensively assessed. HMM Venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors Venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the Venom duct and assigned a biological function, thus pointing to their potential role in Venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups ...
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Large-scale discovery of conopeptides and conoproteins in the injectable Venom of a fish-hunting cone Snail using a combined proteomic and transcriptomic approach
Journal of Proteomics, 2012Co-Authors: Aude Violette, Sebastien Dutertre, Daniel Biass, Reto Stocklin, David Piquemal, Dominique Koua, Fabien Pierrat, Philippe FavreauAbstract:Predatory marine Snails of the genus Conus use Venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable Venom from Conus consors, an indo-pacific fish-hunting cone Snail. By matching MS/MS data against an extensive set of Venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the Venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable Venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone Snail Venom to date, and delineate the major protein families present in the delivered Venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.