The 3-hydroxy-3-methylglutaryl neohesperidosides of hesperetin (brutieridin, 1) and naringenin (melitidin, 2) were isolated and detected from the fruits of bergamot (Citrus bergamia). The structures of these compounds were determined by spectroscopic and chemical methods.

In this article we describe a versatile and straightforward preparative approach to chiral aryl alpha-amino ketones via a Friedel-Crafts-type reaction of stable and enantiomerically pure N-Fmoc protected L-amino acid chlorides with toluene in the presence of aluminum trichloride. The developed methodology provided aryl alpha-amino-p-methylphenyl ketones, which can be obtained and isolated as free bases or recovered as their N-acetyl derivatives, after treatment with acetic anhydride in chloroform at room temperature, subsequent to the Lewis acid induced removal of the 9-fluorenylmethoxycarbonyl protecting group. The Friedel-Crafts-like process and the cleavage of the amino function masking group can selectively be performed since, as verified in all cases, the alpha-aminoacylation step occurred with kinetics that were faster than those required to remove the N-protection. The presented approach was also explored as a facile and useful synthetic tool for the preparation of optically pure ketone di- and tripeptides. These compounds can be obtained in exceptionally overall yields without need of chromatographic purification. Moreover, either aryl alpha-amino ketones or modified di- and tripeptides, in all cases, can be isolated in very high chemical and optical purity without recourse to resolution of diastereomeric mixtures, since the chiralities of the asymmetric amino acid educts were completely conserved throughout the entire process.

The proteic profiling of bovine milk produced by cows with subclinical mastitis was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of raw milk was developed, whereby less complex fractions of proteins were obtained prior to mass spectrometric and SDS-PAGE analysis. The profiles of milk proteins thus obtained could allow the identification of either early markers of the acute phase of mastitis or endogenous peptide of innate immune response. The activity of the endogenous proteases in raw milk produced from each quarter of healthy and mastic cows was therefore assayed over 24-, 48-, 96-, and 216-h incubation at 37 degrees C at both physiological and acid pH. Sequence-specific peptides were identified for each fraction by MS/MS experiments, and all tandem mass spectra were evaluated using MASCOT database searching. The results show a specific proteolytic activity of endogenous enzyme toward beta-casein precursor (P02666), alpha-S2-casein (P02663), alpha-S1-casein (P02662), and kappa-casein (P02668).

Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.

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We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

Quality and safety control and the validation of origin are hot issues in the production of food and its distribution, and are of primary concern to food and agriculture organization. Modern mass spectrometry (MS) provides unique, reliable and affordable methodologies to approach with a high degree of scientificity any problem which may be posed in this field. In this review the contribution of mass spectrometry to food analysis is presented aiming at providing clues on the fundamental role of the basic principles of gas-phase ion chemistry in applied research fields. Applications in proteomics, allergonomics, glycomics, metabolomics, lipidomics, food safety and traceability have been surveyed. The high level of specificity and sensitivity of the MS approach allows the characterization of food components and contaminants present at ultra-trace levels, providing a distinctive and safe validation of the products.

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