IR action spectroscopy shows competitive oxazolone and diketopiperazine formation in peptides depends on peptide length and identity of terminal residue in the departing fragment.

The interplay between the entropically and enthalpically favored products of peptide fragmentation is probed using a combined experimental and theoretical approach. These b2 ion products can take either an oxazolone or diketopiperazine structure. Cleavage after the second amide bond is often a favorable process because the products are small ring structures that are particularly stable. These structures are structurally characterized by action IRMPD spectroscopy and semi-quantified using gas-phase hydrogen-deuterium exchange. The formation of the oxazolone and diketopiperazine has been thought to be largely governed by the identity of the first two residues at the N-terminus of the peptide. We show here that the length of the precursor peptide and identity of the third residue play a significant role in the formation of the diketopiperazine structure in peptides containing an N-terminal asparagine residue. This is additionally the first instance showing an N-terminal residue with an amide side chain can promote formation of the diketopiperazine b2 ion structure.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-adrenoceptor.

BACKGROUND AND PURPOSE: Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs. EXPERIMENTAL APPROACH: We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. KEY RESULTS: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. CONCLUSIONS AND IMPLICATIONS: AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.

Comparison of the fragmentation pattern induced by collisions, laser excitation and electron capture. Influence of the initial excitation.

Collision-induced dissociation, laser-induced dissociation and electron-capture dissociation are compared on a singly and doubly protonated pentapeptide. The dissociation spectrum depends on the excitation mechanism and on the charge state of the peptide. The comparison of these results with the conformations obtained from Monte Carlo simulations suggests that the de-excitation mechanism following a laser or an electron-capture excitation is related to the initial geometry of the peptide.

Ter-body intermediates in the gas phase: reaction of ionized enols with tert-butanol.

In the gas phase, the CH2CHOH.+ enol radical cation 1 as well as its higher homologues CH3CHCHOH.+ 2 and (CH3)2CCHOH.+ 3, undergo exactly the same sequence of reactions with tert-butanol, leading to the losses of isobutene, water and water plus alkene. Fourier transform ion cyclotron resonance (FT-ICR) experiments using labeled reactants as well as ab initio calculations show that independent pathways can be proposed to explain the observed reactivity. For ion 1, taken as the simplest model, the first step of the reaction is formation of a proton bound complex which gives, by a simple exothermic proton transfer, the ter-body intermediate [CH2CHO., H2O, C(CH3)3+]. This complex, which was shown to possess a significant lifetime, is the key intermediate which undergoes three reactions. First, it can collapse to yield tert-butylvinyl ether with elimination of water. Second, by a regiospecific proton transfer, this complex can isomerize into three different ter-body complexes formed of water, isobutene and ionized enol. Within one of these complexes, which does not interconvert with the others, elimination of isobutene leads to the formation of a solvated enol ion. Within the others, a cycloaddition-cycloreversion process can proceed to yield the ionized enol 3 (loss of water and ethylene channel).

Proton affinity and heat of formation of vinyloxy [CH2CHO]· and Acetonyl [CH2COCH3]· radicals.

Frequently found in hydrocarbon oxidation and in the photochemistry of carbonyl compounds, the ß-carbonyl radicals are of interest. The experimental proton affinities of the two title radicals have been determined from proton transfer reactions (as shown) monitored in an FT-ICR mass spectrometer. This led to an estimation of their heats of formation (1: 13±3; 2: -34±3 kJ mol(-1)). Ab initio molecular orbital calculations, up to the G2 level, confirmed these results.

Analysis of alkenes by copper ion chemical ionization gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry

A novel chemical ionization/fast atom bombardment (CI/FAB) source was used to analyse alkenes by chemical ionization mass spectrometry (CI-MS) using copper ions as the ionizing agent. The Cu(+)-CI mass spectra showed abundant pseudomolecular adduct ions [alkene-Cu](+) and characteristic fragment ions. Mass-analysed ion kinetic energy spectroscopy was used to study the product ions resulting from the decomposition of adduct ions and to eliminate background interferences derived from the copper ions. The major fragmentations permitted the localization of double bonds and minor fragments allowed the differentiation of alkene isomers. The CI/FAB source was coupled to a gas chromatograph and simple and complex mixtures of octene isomers were analysed by gas chromatography (GC)/Cu(+)-CI-MS and GC/Cu(+)-CI-MS/MS. Copyright 1999 John Wiley & Sons, Ltd.

A comparison of matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography electrospray ionization mass spectrometry methods for the analysis of crude tarantula venoms in the Pterinochilus group.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed-phase chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinus group. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI-TOFMS and ESI-MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI-MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution.

Trace determination of glycols by HPLC with UV and electrospray ionization mass spectrometric detections.

A high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is developed for trace determination of glycols (ethylene glycol, 1,2- and 1,3-propylene glycols, and 2,3-butylene glycol) in water after derivatization with benzoyl chloride. Benzoyl esters of glycols are separated by microcolumn reversed-phase HPLC. Sensitivity and linearity of UV detection at 237 nm is compared with electrospray ionization mass spectrometric (ESI-MS) detection using selected ion monitoring. Limits of detection (LOD) and quantitation (LOQ) for UV detection are 1 and 2 mg/L, respectively. For ESI-MS detection, LOD and LOQ are in the ranges 10-25 and 20-50 µg/L, respectively. LOD obtained by ESI-MS for the determination of glycols is improved by 2-3 orders of magnitude in comparison to previously published methods. The effect of the structure of isomeric glycols on their electrospray mass spectra is discussed. The method has been applied for the determination of glycols in aqueous matrixes containing high concentrations of salts occurring in nuclear waste disposal treatment.