No abstract

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose.

High-resolution mass spectrometry-based peptidomics has been used to characterize several components in electro-stimulated skin secretions of the endemic Mexican frog Pachymedusa dacnicolor. Peptide mass screening performed in an Orbitrap-XL mass spectrometer showed that P. dacnicolor skin secretions possess 194 different components with molecular masses ranging mainly from 500 to 6,000 Da. Dozens of molecules were partially sequenced including two novel protease inhibitors. Additionally, one posttranslationally modified bradykinin and two novel dermaseptin-like antimicrobial peptides were fully sequenced. The novel peptide named here DMS-DA5 was fully characterized and showed potent antibacterial activity against various bacteria such as Escherichia coli, Bacillus subtilis, Salmonella enterica serovar typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations from 3.10 to 25.0 microM.

Venom glands and soluble venom from the Mexican scorpion Centruroides limpidus (Karsch, 1879) were used for transcriptomic and proteomic analyses, respectively. An RNA-seq was performed by high-throughput sequencing with the Illumina platform. Approximately 80 million reads were obtained and assembled into 198,662 putative transcripts, of which 11,058 were annotated by similarity to sequences from available databases. A total of 192 venom-related sequences were identified, including Na+ and K+ channel-acting toxins, enzymes, host defense peptides, and other venom components. The most diverse transcripts were those potentially coding for ion channel-acting toxins, mainly those active on Na+ channels (NaScTx). Sequences corresponding to β- scorpion toxins active of K+ channels (KScTx) and λ-KScTx are here reported for the first time for a scorpion of the genus Centruroides. Mass fingerprint corroborated that NaScTx are the most abundant components in this venom. Liquid chromatography coupled to mass spectometry (LC-MS/MS) allowed the identification of 46 peptides matching sequences encoded in the transcriptome, confirming their expression in the venom. This study corroborates that, in the venom of toxic buthid scorpions, the more abundant and diverse components are ion channel-acting toxins, mainly NaScTx, while they lack the HDP diversity previously demonstrated for the non-buthid scorpions. The highly abundant and diverse antareases explain the pancreatitis observed after envenomation by this species.

Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue certainly could bring in much more information for understanding this molecular process and its biochemical consequences. Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted 253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/ MS were used for "de novo" sequencing of 111 different peptides and the unequivocal identification of fifteen proteins including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high antibacterial, antiparasitic and cytotoxic activities.

Centruroides possanii is a recently discovered species of “striped scorpion” found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.