The semisynthesis of octadeutero-PheB1-octadeutero-ValB2]-procine insulin and its characterization by mass spectrometry

Insulin analogues labelled with stable isotopes (e.g. deuterium, 18O, 15N, etc.) are authentic (the native structure is rigorously maintained), non-radioactive (preferred for injection into man) and can easily be distinguished from endogenous insulin by mass spectrometry by virtue of their molecular masses. Appropriate combinations of amino-protecting groups (methylsulphonylethyloxycarbonyl and t-butoxy carbonyl), Edman degradation and chemical coupling were used to produce [octadeutero-PheB1]-porcine insulin and [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin. The analogues were characterized by electrospray ionization mass spectrometry. Standard mixtures of labelled and unlabelled insulins were successfully studied by mass spectrometry. Isotope dilution mass spectrometry could therefore provide a useful direct measure of insulin under true physiological conditions, without many of the drawbacks of existing methods. In this regard, the analogue with 16 deuteriums was more suitable than the octadeuterated analogue, since the greater mass difference between the labelled and unlabelled forms enabled a lower mass spectrometric resolution to be used, resulting in higher sensitivit

The mitochondrial genome of the venomous cone snail conus consors

Cone snails are venomous predatory marine neogastropods that belong to the species-rich superfamily of the Conoidea. So far, the mitochondrial genomes of two cone snail species (Conus textile and Conus borgesi) have been described, and these feed on snails and worms, respectively. Here, we report the mitochondrial genome sequence of the fish-hunting cone snail Conus consors and describe a novel putative control region (CR) which seems to be absent in the mitochondrial DNA (mtDNA) of other cone snail species. This possible CR spans about 700 base pairs (bp) and is located between the genes encoding the transfer RNA for phenylalanine (tRNA-Phe, trnF) and cytochrome c oxidase subunit III (cox3). The novel putative CR contains several sequence motifs that suggest a role in mitochondrial replication and transcription

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry

The production of semisynthetic human insulin for therapeutic purposes is of considerable importance. During trypsin-catalysed transformation of pig insulin into an ester of insulin of human sequence, the alanyl residue at position B30 is removed and replaced with an esterified residue of threonine. We have carried out this transformation in a medium enriched in 18OH2 and studied the product by MS. In contrast to a previous report, we find that incorporation of label into the B29−B30 peptide bond occurs during the transformation with threonine methyl ester in aqueous N, N-dimethylacetamide. Quantitative data are presented and the implications of these findings are discusse

Recruitment of Glycosyl Hydrolase Proteins in a Cone Snail Venomous Arsenal: Further Insights into Biomolecular Features of Conus Venoms

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms (“injectable venom” stands for the venom variety obtained by milking of the snails. This is in contrast to the “dissected venom”, which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms

Future perspectives of venoms for drug discovery

Venoms have been regarded as an interesting source for screening difficult-to-address pharmaceutical targets like ion channels, transmembrane or circulating proteins for quite some time. Moreover, recent developments open up new opportunities to better exploit well-structured toxins - so called “mini-proteins” that we also like to refer to as “picologics” - in a more general way for lead discovery and drug development. In particular, progress in throughput and the first results from the next generation of genome/transcriptome analyses and systems biology investigations widen the scope of an integrated understanding of global transcriptional activity in venom glands. As a result, natural products from venoms are thought to represent a sustainable source of molecules for addressing a wider range of targets, and thus enable a more systematic approach to the identification of new peptide therapeutic candidates

Venom composition and strategies in spiders: is everything possible?

This review on all spider venom components known by the end of 2010 bases on 1618 records for venom compounds from 174 spider species (= 0.41% of all known species) belonging to 32 families (= 29% of all existing spider families). Spiders investigated for venom research are either big (many mygalomorph species, Nephilidae, Ctenidae and Sparassidae) or medically important for humans (e.g. Loxosceles or Latrodectus species). Venom research widely ignored so far the two most species-rich families (Salticidae and Linyphiidae) and strongly neglected several other very abundant families (Araneidae, Lycosidae, Theridiidae, Thomisidae and Gnaphosidae). We grouped the known 1618 records for venom compounds into six categories: low molecular mass compounds (16 % of all compounds), acylpolyamines (11 %), linear peptides (6 %), cysteine-knotted mini-proteins (60 %), neurotoxic proteins (1 %) and enzymes (6 %). Low molecular mass compounds are known from many spider families and contain organic acids, nucleosides, nucleotides, amino acids, amines, polyamines, and some further substances, many of them acting as neurotransmitters. Acylpolyamines contain amino acids (Araneidae and Nephilidae) or not (several other families) and show a very high diversity within one species. Linear peptides, also called cytolytic, membranolytic or antimicrobial, exert a highly specific structure and are so far only known from Ctenidae, Lycosidae, Oxyopidae and Zodariidae. Cysteine-knotted mini-proteins represent the majority of venom compounds because research so far focused on them. They probably occur in most but not all spider families. Neurotoxic proteins so far are only known from theridiid spiders. Enzymes had been neglected for some time but meanwhile it becomes obvious that they play an important role in spider venoms. Sixteen enzymes either cleave polymers in the extracellular matrix or target phospholipids and related compounds in membranes. The overall structure of these compounds is given and the function, as far as it is known, is described. Since several of these component groups are presented in one average spider venom, we discuss the known interactions and synergisms and give reasons for such a functional redundancy. We also discuss main evolutionary pathways for spider venom compounds such as high variability among components of one group, synergistic interactions between cysteine-knotted mini-proteins and other components (low molecular mass compounds and linear peptides), change of function from ion-channel acting mini-proteins to cytolytic effects and replacement of mini-proteins by linear peptides, acylpolyamines, large proteins or enzymes. We also add first phylogenetic considerations

Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: Case applications with single arthropod specimen

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Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry

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Data from: Predicting function from sequence in a large multifunctional toxin family

Venoms contain active substances with highly specific physiological effects and are increasingly being used as sources of novel diagnostic, research and treatment tools for human disease. Experimental characterisation of individual toxin activities is a severe rate-limiting step in the discovery process, and in-silico tools which allow function to be predicted from sequence information are essential. Toxins are typically members of large multifunctional families of structurally similar proteins that can have different biological activities, and minor sequence divergence can have significant consequences. Thus, existing predictive tools tend to have low accuracy. We investigated a classification model based on physico-chemical attributes that can easily be calculated from amino-acid sequences, using over 250 (mostly novel) viperid phospholipase A2 toxins. We also clustered proteins by sequence profiles, and carried out in-vitro tests for four major activities on a selection of isolated novel toxins, or crude venoms known to contain them. The majority of detected activities were consistent with predictions, in contrast to poor performance of a number of tested existing predictive methods. Our results provide a framework for comparison of active sites among different functional sub-groups of toxins that will allow a more targeted approach for identification of potential drug leads in the future

Advances in venomics

The term "venomics" was coined to describe the global study of venom and venom glands, targeting comprehensive characterization of the whole toxin profile of a venomous animal by means of proteomics, transcriptomics, genomics and bioinformatics studies. This integrative approach is supported by the rapid evolution of protein, RNA and DNA sequencing techniques, as well as databases, knowledge-bases and biocomputing algorithms. The aim of this review is to illustrate advances in the field of venomics during the last decade, addressing each step of the procedure, from sample collection to data treatment. A special focus is made on new perspectives for a better understanding of the venomous function and for fostering the discovery of new venom-derived drug candidates