Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification. Copyright (C) 2002 John Wiley Sons, Ltd.

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH2 (MW 1611.98Da) and IDWLKLGKMVMDVL-NH2 (MW 1658.98Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of poly-morphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging. Copyright (C) 2004 John Wiley Sons, Ltd.

Proteomic Characterization of the Hyaluronidase (EC 3.2.1.35) from the Venom of the Social Wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins from the barks of Hancornia speciosa

Direct flow injection electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) was used to investigate the polyphenolic compounds present in an infusion from the barks of Hancornia speciosa Gom. (Apocynaceae), a native Brazilian plant popularly known as 'mangabeira', used as a source of nutrition and against gastric disorders. After a simple sample filtration pretreatment the characteristic fingerprint of the infusion was performed in negative ion ESI mode in a few minutes. At low capillary-voltage activation, the deprotonated molecules ([M-H](-)) were observed and using collision-induced dissociation the product ion spectra showed the presence of a homologous series of B-type proanthocyanidins, as well as another series containing their respective C-glycosylated derivatives, with a degree of polymerization from 1 up to 6 units of interlinked catechins. Therefore, direct flow injection allowed us to identify the key compounds without preparative isolation of the components. Copyright (C) 2007 John Wiley & Sons, Ltd.

Dissociation between the circulating renin-angiotensin system and angiotensin II receptors in central losartan-induced hypertension

Losartan, an AT1 angiotensin II (ANG II) receptor non-peptide antagonist, induces an increase in mean arterial pressure (MAP) when injected intracerebroventricularly (icv) into rats. The present study investigated possible effector mechanisms of the increase in MAP induced by icv losartan in unanesthetized rats. Male Holtzman rats (280-300 g, N = 6/group) with a cannula implanted into the anterior ventral third ventricle received an icv injection of losartan (90 µg/2 µl) that induced a typical peak pressor response within 5 min. In one group of animals, this response to icv losartan was completely reduced from 18 ± 1 to 4 ± 2 mmHg by intravenous (iv) injection of losartan (2.5-10 mg/kg), and in another group, it was partially reduced from 18 ± 3 to 11 ± 2 mmHg by iv prazosin (0.1-1.0 mg/kg), an alpha1-adrenergic antagonist (P<0.05). Captopril (10 mg/kg), a converting enzyme inhibitor, injected iv in a third group inhibited the pressor response to icv losartan from 24 ± 3 to 7 ± 2 mmHg (P<0.05). Propranolol (10 mg/kg), a ß-adrenoceptor antagonist, injected iv in a fourth group did not alter the pressor response to icv losartan. Plasma renin activity and serum angiotensin-converting enzyme activity were not altered by icv losartan in other animals. The results suggest that the pressor effect of icv losartan depends on angiotensinergic and alpha1-adrenoceptor activation, but not on increased circulating ANG II.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations. © 2012 Elsevier B.V.

Role of the range of the dipole function in the classical dynamics of molecular dissociation

The dissociation dynamics of heteronuclear diatomic molecules induced by infrared laser pulses is investigated within the framework of the classical driven Morse oscillator. The interaction between the molecule and the laser field described in the dipole formulation is given by the product of a time-dependent external field with a position-dependent permanent dipole function. The effects of changing the spatial range of the dipole function in the classical dissociation dynamics of large ensembles of trajectories are studied. Numerical calculations have been performed for distinct amplitudes and carrier frequencies of the external pulses and also for ensembles with different initial energies. It is found that there exist a set of values of the dipole range for which the dissociation probability can be completely suppressed. The dependence of the dissociation on the dipole range is explained through the examination of the Fourier series coefficients of the dipole function in the angle variable of the free system. In particular, the suppression of dissociation corresponds to dipole ranges for which the Fourier coefficients associated with nonlinear resonances are null and the chaotic region in the phase space is reduced to thin layers. In this context, it is shown that the suppression of dissociation of heteronuclear molecules for certain frequencies of the external field is a consequence of the finite range of the corresponding permanent dipole. © 2013 American Physical Society.

Quantum-classical correspondence and the role of the dipole function in molecular dissociation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)