Analysis of sulfated peptides from the skin secretion of the Pachymedusa dacnicolor frog using IMAC-Ga enrichment and high-resolution mass spectrometry

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.     

Development of small molecule inhibitors and probes of human SUMO deconjugating proteases

Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.     

Sulfonamide carbazole receptors for anion recognition

Carbazole-based receptors functionalized with two sulfonamide groups have been synthesized and their properties as anion receptors have been evaluated. The receptor with bis(trifluoromethyl)aniline groups has shown a very high affinity for halide ions, especially remarkable as only two hydrogen bonds are formed in the complexes. (1)H NMR and fluorescence titrations have been carried out and binding constants up to 7.9 × 10(6) M(-1) have been reached. X-ray structures have been obtained and a modelling study has shown the possible reasons for the large affinity of these compounds for halide anions.     

Unexpected phenol production from arylboronic acids under palladium-free conditions; organocatalyzed air oxidation

An intriguing class of quinones that efficiently catalyze the air oxidation (overall hydroxylation) of arylboronic acids to the corresponding phenol is reported. Autocatalysis in the parent system is particularly efficient and leads to rapid, quantitative synthesis of quinones such as 4 from boronic acid 1 at room temperature using air as stoichiometric oxidant. The efficiency results from a balance between two-stage conjugate addition and migration with each step driven by aromatization of a naphthalene fragment.     

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6-monoacetylmorphine in vitreous humor

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 μL.     

Development and validation of an HPLC method for the determination of ten sulfonamide residues in milk according to 2002/657/EC

A HPLC method with diode-array detection, at 265 nm, was developed and validated for the determination of ten sulfonamides (SAs): sulfadiazine (SDZ), sulfathiazine (STZ), sulfamethoxine (SMTH), sulfamethizole (SMZ), sulfamethoxypyridazine (SMPZ), sulfamonomethoxine (SMMX), sulfamethoxazole (SMXZ), sulfisoxazole (SIX), sulfadimethoxine (SDMX), and sulfaquinoxaline (SQX) in milk. A mixture of ethyl acetate, n-hexane, and isopropanol was used for the extraction of target analytes from milk. The mobile phase, a mixture of 0.1% v/v formic acid, CH(3) CN, and CH(3) OH was delivered to the analytical column under a gradient program. The procedure was validated according to the European Union regulation 2002/657/EC in terms of selectivity, stability, decision limit, detection capability, accuracy, and precision. Mean recoveries of sulfonamides from milk samples spiked at three concentration levels (0.5×MRL, 1×MRL, and 1.5×MRL) (MRL, maximum residue level) were 93.9-115.9% for SDZ, 97.8-102.9% for STZ, 94.6-107.0% for SMTH, 98.3-111.5% for SMZ, 95.3-108.4% for SMPZ, 97.9-106.0% for SMMX, 97.6-111.3% for SMXZ, 94.3-104.6% for SIX, 96.4-109.1% for SDMX, and 98.2-111.2% for SQX. All RSD values were lower than 8.8%. The decision limits CCa calculated by spiking 20 blank milk samples at MRL (100 μg/kg) ranged from 101.61 to 106.84 μg/kg, whereas the detection capability CCb ranged from 105.64 to 119.01 μg/kg.     

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.     

Axiomatisability problems for <Emphasis Type="Italic">S</Emphasis>-posets

Let \(\mathcal{C}\) be a class of ordered algebras of a given fixed type τ. Associated with the type is a first order language L _τ , which must also contain a binary predicate to be interpreted by the ordering in members of \(\mathcal{C}\). One can then ask the question, when is the class \(\mathcal{C}\) axiomatisable by sentences of L _τ ? In this paper we will be considering axiomatisability problems for classes of left S-posets over a pomonoid S (that is, a monoid S equipped with a partial order compatible with the binary operation). We aim to determine the pomonoids S such that certain categorically defined classes are axiomatisable. The classes we consider are the free S-posets, the projective S-posets and classes arising from flatness properties. Some of these cases have been studied in a recent article by Pervukhin and Stepanova. We present some general strategies to determine axiomatisability, from which their results for the classes of weakly po-flat and po-flat S-posets will follow. We also consider a number of classes not previously examined.     

Simultaneous Determination of 1,4‐Benzodiazepines and Tricyclic Antidepressants in Saliva after Sequential SPE Elution by the Same HPLC Conditions

A new method has been described to determine both benzodiazepines (six) and tricyclic antidepressants (four) simultaneously in saliva by HPLC with a UV detector set at 240 nm using cholchicine as the internal standard. A careful specific sequential solid‐phase elution was optimized and performed to elute benzodiazepines using a mixture of methanol‐acetonitrile (1:1 v/v) followed by the elution of tricyclic antidepressants with methanol. Separation of the compounds was performed on a Kromasil column (250 × 4 mm, 5 μm) by a gradient eluents consisting of 0.05 M CH₃COONH₄‐acetonitrile‐methanol (55:15:30 v/v/v). The results were linear for both benzodiazepines and tricyclic antidepressants up to 20 ng μL^‐1 with the correlation coefficients greater than 0.998. The sensitivity limits, LOD and LOQ were 0.08‐0.34 ng μL^‐1 and 0.28‐1.13 ng μL^‐1, respectively. The method is simple, fast and reliable with good specificity and sensitivity, will be suitable for use in a clinical setting, where there is a concomitant use of 1,4‐benzodiazepines and tricyclic antidepressants.     

Peculiarities of the complexation of copper and silver adducts of a 3,5-bis(trifluoromethyl)pyrazolate ligand with organoiron compounds

Interaction of the copper, {[3,5-(CF(3))(2)Pz]Cu}(3), and silver, {[3,5-(CF(3))(2)Pz]Ag}(3), macrocycles [3,5-(CF(3))(2)Pz = 3,5-bis(trifluoromethyl)pyrazolate] with cyclooctatetraeneiron tricarbonyl, (cot)Fe(CO)(3), was investigated by IR and NMR spectroscopy for the first time. The formation of 1:1 complexes was observed at low temperatures in hexane. The composition of the complexes (1:1) and their thermodynamic characteristics in hexane and dichloromethane were determined. The π-electron system of (cot)Fe(CO)(3) was proven to be the sole site of coordination in solution and in the solid state. However, according to the single-crystal X-ray data, the complex has a different (2:1) composition featuring the sandwich structure. The complexes of ferrocene with copper and silver macrocycles have a columnar structure (X-ray data).