Amine-functionalized sol-gels were investigated for the enrichment and purification of phosphopeptides from digested protein mixture solutions. Tetramethylorthosilicate (TMOS) and N'[3-(trimethoxysilyl)-propyl]-diethylenetriamine (TPDA) were used in a 1:1 mole ratio in the production of amine-functionalized sol-gels. The sol-gel network was then used for phosphopeptide enrichment. Phosphopeptide enrichment onto the synthesized amine-functionalized sol-gels was performed using an enolase digested peptide mixture, a β-casein digested peptide mixture, as well as these digested peptide mixtures contaminated 50-fold with bovine serum albumin (BSA). Moreover, phosphopeptide enrichment was successfully performed using nonfat milk as a highly contaminated and complex material. In each phosphopeptide enrichment and purification process, only phosphopeptides were enriched and separated from the other digested peptides. Phosphopeptides were adsorbed onto the amine-functionalized sol-gels at pH 4.0 and eluted at pH 1.0 using trifluoroacetic acid (TFA). For phosphopeptide analysis by MALDI-MS, a 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid was used to overcome the degradation of phosphopeptides and provide high intensity phosphopeptide protonated molecular ion signal intensities. It was also found that phosphopeptide detection limits were improved to approximately 10 femtomoles. For rapid and specific phosphopeptide enrichment and purification, sol-gel materials were placed in a 10 μL pipet tip with glass wool on either side. Phosphopeptide enrichment from digested peptide mixtures was performed in a very short time (less than 1 min) at subpicomole levels using this novel lab-in-a-pipet-tip approach.

A new tantalum-based sol-gel material was synthesized using a unique sol-gel synthesis pathway by PEG incorporation into the sol-gel structure without performing a calcination step. This improved its chemical and physical properties for the high capacity and selective enrichment of phosphopeptides from protein digests in complex biological media. The specificity of the tantalum-based sol-gel material for phosphopeptides was evaluated and compared with tantalum(V) oxide (Ta2O5) in different phosphopeptide enrichment applications. The tantalum-based sol-gel and tantalum(V) oxide were characterized in detail using FT-IR spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), and also using a surface area and pore size analyzer. In the characterization studies, the surface morphology, pore volume, crystallinity of the materials and PEG incorporation into the sol-gel structure to produce a more hydrophilic material were successfully demonstrated. The X-ray diffractograms of the two different materials were compared and it was noted that the broad signals of the tantalum-based sol-gel clearly represented the amorphous structure of the sol-gel material, which was more likely to create enough surface area and to provide more accessible tantalum atoms for phosphopeptides to be easily adsorbed when compared with the neat and more crystalline structure of Ta2O5. Therefore, the phosphopeptide enrichment performance of the tantalum-based sol-gels was found to be remarkably higher than the more crystalline Ta2O5 in our studies. Phosphopeptides at femtomole levels could be selectively enriched using the tantalum-based sol-gel and detected with a higher signal-to-noise ratio by matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS). Moreover, phosphopeptides in a tryptic digest of non-fat bovine milk as a complex real-world biological sample were retained with higher yield using a tantalum-based sol-gel. Additionally, the sol-gel material was packed into a standard syringe (0.5 mL) to enhance the ease of use of the sol-gel material and for the elimination of additional mixing and separation procedures during the adsorption, washing and elution steps of the enrichment procedure. It was found that up to 28 phosphopeptides in milk digest were easily detectable by MALDI-MS at femtomole levels (around 20 fmol) using the microextraction syringe within less than one minute.

Novel hydrazones based on (-)-carvone were synthesized via condensation of terpenoid with 4-R-phenoxyacetic acid hydrazides. The structure of target compounds was established by FT-IR, Raman, 1 H-NMR and 13 C-NMR spectral analysis, FAB/ESI mass spectrometry. (-)-Carvone hydrazones were proven to exist as Z/E geometrical isomers about C=N bond using ion mobility-tandem mass spectrometry (IM-MS/MS). Single crystal X-ray diffraction study was applied to determine molecular and crystal structure of compound 3e. Hydrazones 3a-3e were evaluated as potential anticonvulsant agents after their oral administration against maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in mice. Analgesic activity of compounds was investigated by topical application on models of capsaicin and AITC-induced pain. The present findings indicate that (-)-carvone derivatives afforded seizure protection both at short (1 h) and long (24 h) time period by blocking electroshock-and chemical-induced convulsions.Hydrazones binding to TRPA1/TRPV1 ion channels was proposed as possible mechanism explaining significant analgesic effect of compounds.

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This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.