Rapid ligand fishing for identification of acetylcholinesterase-binding peptides in snake venom reveals new properties of dendrotoxins.

Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino-modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity-based ligand-fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography-mass spectrometry (nanoLC-MS) was used for the analysis of trapped peptides. Using enzyme-functionalized beads, the ligand-fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin-II) and five non-binders (mambalgin-1, angiotensin-II, bradykinin, cardiotoxin and α-bungarotoxin). As proof of concept, snake venom samples spiked with fasciculin-II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 µg/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp.), which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano-LC-MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE-binding affinity as dendrotoxin-I, a pre-synaptic neurotoxin previously not known to interact with AChE. Co-incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilize AChE.

Direct injection of native aqueous matrices by achiral-chiral chromatography ion trap mass spectrometry for simultaneous quantification of pantoprazole and lansoprazole enantiomers fractions.

A two-dimensional liquid chromatography system coupled to ion-trap tandem mass spectrometer (2DLC-IT-MS/MS) was employed for the simultaneous quantification of pantoprazole and lansoprazole enantiomers fractions. A restricted access media of bovine serum albumin octyl column (RAM-BSA C(8)) was used in the first dimension for the exclusion of the humic substances, while a polysaccharide-based chiral column was used in the second dimension for the enantioseparation of both pharmaceuticals. The results described here show good selectivity, extraction efficiency, accuracy, and precision with detection limits of 0.200 and 0.150 µg L(-1) for the enatiomers of pantoprazole and lansoprazole respectively, while employing a small amount (1.0 mL) of native water sample per injection. This work reports an innovative assay for monitoring work, studies of biotic and abiotic enantioselective degradation and temporal changes of enantiomeric fractions.

A column-switching method for quantification of the enantiomers of omeprazole in native matrices of waste and estuarine water samples.

This work reports the use of a two-dimensional liquid chromatography (2D-LC) system for quantification of the enantiomers of omeprazole in distinct native aqueous matrices. An octyl restricted-access media bovine serum albumin column (RAM-BSA C(8)) was used in the first dimension, while a polysaccharide-based chiral column was used in the second dimension with either ultraviolet (UV-vis) or ion-trap tandem mass spectrometry (IT-MS/MS) detection. An in-line configuration was employed to assess the exclusion capacity of the RAM-BSA columns to humic substances. The excluded macromolecules had a molecular mass in the order of 18 kDa. Good selectivity, extraction efficiency, accuracy, and precision were achieved employing a very small amount (500 microL or 1.00 mL) of native water sample per injection, with detection limits of 5.00 microg L(-1), using UV-vis, and 0.0250 microg L(-1), using IT-MS/MS. The total analysis time was only 35 min, with no time spent on sample preparation. The methods were successfully applied to analyze a series of waste and estuarine water samples. The enantiomers were detected in an estuarine water sample collected from the Douro River estuary (Portugal) and in an influent sample from the wastewater treatment plant (WWTP) of São Carlos (Brazil). As far as we are concerned, this is the first report of the occurrence of (+)-omeprazole and (-)-omeprazole in native aqueous matrices.

Resolution and absolute configuration assignment of a natural racemic chromane from Peperomia obtusifolia (Piperaceae).

The resolution of the natural racemic chromane 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3''-methyl-2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2H-1-benzopyran-6-carboxylic acid (1) isolated from the leaves of Peperomia obtusifolia has been accomplished using stereoselective HPLC. The absolute configuration of the resolved enantiomers was determined by the analysis of optical rotations and CD spectra. The finding of a racemic mixture instead of an enantiomerically pure metabolite raises questions about the final steps in the biosynthesis of this class of natural products, suggesting that the intramolecular chromane ring formation step may not be enzymatically controlled at all in P. obtusifolia.