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The study of unsubstituted and disubstituted β-cyclodextrins (β-CDs) by ESI-mass spectrometry is reported, applying a cone-induced fragmentation in the presence of a twofold excess of sodium chloride, in order to gain information about the fragmentation of the different regioisomers. On the basis of the fragmentation pattern observed for the unsusbstituted β-CD, a statistical model shows that the fragments generated by every regioisomer of a disubstituted CD (AB, AC, and AD) are expected to differ in their relative intensity and, therefore, they can be used for correctly identifying the three different regioisomers. The model was tested on the three regioisomeric (AB, AC, and AD) diamino-β-CDs and ditosyl-β-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-β-CD, allowing in every case through statistical analysis of the fragmentation pattern the correct assignment of every regioisomer on the basis of an ESI mass spectrum (single quadrupole analyzer, high cone voltage) of the pure compounds. The absolute intensities of the fragmentation peaks were voltage-dependent but their ratios was voltage-independent, indicating that no mass bias in peak ratios is introduced by the analyzer. Given the fast time of analysis and its general applicability, independently from the substituents, we propose our method as an easy way to identify the regioisomers of disubstituted β-CDs. [Copyright &y& Elsevier]

Resistance of gluten to gastrointestinal digestion is involved in immune-mediated adverse reactions to wheat, since several peptides produced by the incomplete digestion are able to trigger, in predisposed individuals, the immune response responsible, for instance, of celiac disease (CD) and other adverse reactions. Even if several peptides have been identified, an exhaustive description of the peptidome generated by wheat digestion is lacking. To this end, in the present work, durum wheat proteins were fractionated, digested, and then subjected to various proteomic techniques, including single stage and multiple stage mass spectrometry (MS) (SDS-PAGE, UPLC/ESI-MS, UPLC/ESI-MS/MS, and LTQ-Orbitrap). Based on SDS-PAGE, although proteins were severely degraded after in vitro gastrointestinal digestion, some differences were observed among protein profile of the different digests. Through untargeted UPLC techniques, 227 peptide sequences were identified, with only few sequences shared by the different digests. In particular, 9 gluten peptides involved in CD were identified. Based on target proteomic, the quantification of these peptides revealed significant (p ≤ 0.05) differences among the different extracts. Taken together, all the proteomic tools confirmed that gluten digestion is closely related to the matrix regardless of wheat genotype. [ABSTRACT FROM AUTHOR]

Durum wheat ( Triticum turgidum L. subsp. durum (Desf.) Husn) is a major food source in Mediterranean countries since it is utilized for the production of pasta, leavened and unleavened breads, couscous, and other traditional foods. The technological and nutritional properties of durum wheat semolina depend mainly on the type of gluten proteins and on their amount, which is a genotype- and environment-dependent trait. Gluten proteins are also responsible for celiac disease (CD), an autoimmune enteropathy with a prevalence of about 0.7-2% in the human population. At this purpose, two Italian durum wheat cultivars, Saragolla and Cappelli, currently used for monovarietal pasta, were chosen to compare (i) the reserve and embryo proteome, (ii) the free and bound phenolics, antioxidant activity, and amino acid composition, and (iii) the content of immunogenic peptides produced after a simulated gastrointestinal digestion. The results obtained from 2 years of field cultivation on average showed a higher amount of gluten proteins, amino acids, and immunogenic peptides in Cappelli. Saragolla showed a higher abundance in bound phenolics, antioxidant enzymes, and stress response proteins in line with its higher antioxidant activity. However, the impact of the year of cultivation, largely depending on varying rainfall regimes through the wheat growth cycle, was significant for most of the parameters investigated. Differences in technological and nutritional characteristics observed between the two cultivars are discussed in relation to the influence of genetic and environmental factors.

The knowledge of pectin esterification degree is of primary importance to predict gelling and other properties of pectin from different sources. This paper reports the development of a simple and rapid (1)H NMR-based method for the simultaneous quantitative determination of methylation, acetylation, and feruloylation degree of pectin isolated from various food sources. Pectin esters are hydrolyzed in NaOH/D2O, and the obtained methanol, acetic acid, and ferulic acid are directly measured by (1)H NMR. High accuracy, repeatability, and reproducibility of the method were obtained, and the analysis time is reduced as compared to conventional chromatography- or titration-based methods.

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

The synthesis of C5 linked uracil dimers was carried out according to a model developed in order to bind adenine in DNA. N1-Alkylated uracil derivatives were synthesized from isoorotic acid (uracil-5-carboxylic acid) or thymine. The carboxylic acid derivatives were condensed with diamines in order to produce dimeric compounds or with monoamines in order to obtain reference monomeric compounds. Some of the derivatives, in particular the uracil dimers, showed antiproliferative and erythroid differentiation induction properties towards human chronic myelogenous leukemia K562 cells, thus indicating that these compounds could represent a new class of drugs useful for the development of antitumor therapy based on the ability to induce terminal differentiation.

Lipid transfer proteins (LTPs) are a family of low molecular mass (7-9 kDa) polypeptides, the members of which share 35-95% sequence homology. These proteins are widely distributed throughout the plant kingdom and are receiving attention for their biochemical characteristics and biological activity. LTPs are indeed studied in different research fields varying from allergy to food technology, and numerous molecules belonging to this class are progressively being identified and investigated. Proteins from pomegranate juice were fractioned by cation exchange chromatography and analyzed by SDS-PAGE. Two proteins were identified as putative LTPs on the basis of their molecular weights and their electrophoretic behaviors under reducing and nonreducing conditions. Finally, proteins were purified and characterized by mass spectrometry. This analysis confirmed that the two polypeptides are LTPs on the basis of an amino acid sequence common to LTPs from other plant sources and cysteine content. The two proteins, named LTP1a and LTP1b, showed similar molecular masses but different immunological profiles when immunodetected with rabbit antibodies specific for Pru p 3 and human IgE from a patient suffering from pomegranate allergy. The demonstration of the existence of two immunologically unrelated LTPs in pomegranate confirms the variability and the complexity of the plant LTP family. This should be taken into account when the role of these proteins as elicitors of allergies to fruits is investigated and could help to explain the contradictory literature data on pomegranate allergy.

PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

The effect of the dry-curing processing time on the release of oligopeptides and amino acids was evaluated with 158 Parma hams subdivided into three groups: (1) traditional processing (450 days); (2) extended processing (570 days); and (3) extended aging (690 days). Most of the oligopeptides and free amino acids detected increased up to the last deadline (690 days); a sharp increase of peptides below 400 Da was the main change in most aged hams. In particular, gamma-glutamyl dipeptides showed a remarkable increase during ham extended aging, acting like permanent taste-active compounds, being unsuitable for further enzymatic breakdown. The pH of fresh hams showed negative relationships (P < 0.001) with most peptides. With regard to free amino acids, the pattern was modified by different processing lengths, together with their taste categories, so that the amino acids having monosodium glutamate-like and bitter tastes were enhanced in more aged hams.

Sixteen samples of Grana Padano cheese aged from 2 to 33 months were analyzed by HPLC-MS. The extraction process involved the use of diluted HCl, thus avoiding a strong deproteinizing agent (TCA), and allowing to maintain in solution also very lipophilic peptides. The molecular mass of the most abundant peptides were determined by electrospray ionization (ESI) mass spectrometry. A new method was developed based on the small fragmentation peaks arising from in-source fragmentation and from software analysis of the known casein sequences, which in many cases allowed the direct on-line identification of the oligopeptide sequences. Several new peptides never previously reported were identified, some of which containing bioactive sequences, consistently with what was described in the literature. Semiquantification of peptides at the different stages of aging was also performed by using a suitable internal standard, providing new insights into the evolution of the oligopeptide fraction during aging.