A simple software, to be used as an aid in the identification of non-tryptic peptides based on low resolution (3D-ion trap) tandem (MS/MS) and sequential (MS3) mass spectrometry data, is presented. The program, named INSPIRE (Identification of Non-tryptic peptide Sequences based on Product Ions m/z ratios and RElative abundances), provides alternative rankings for the several candidate sequences usually arising from protein database searches when non-tryptic peptides are involved and only low resolution MS/MS data are available. The rankings, based on parameters related to m/z ratios and relative abundances of experimental product ions matching with predicted ones, can be exploited to reduce the number of candidates to be included in subsequent data processing based on MS3 measurements. The latter usually represents a mandatory step towards a reliable peptide identification when high resolution MS/MS data are not accessible. Sets of peptide sequences arising from MS/MS-based database searches for 63 previously identified non-tryptic peptides (all generated from milk proteins) were exploited to check the INSPIRE performance. It was found that, if retrieved among candidates after the database search, the correct sequence was always ranked among the first 10 ones when parameters calculated by INSPIRE were adopted for discrimination purposes. Under the same conditions the ranks provided by popular database search programs were significantly worse in a remarkable number of cases.

The aim of the present study was to provide experimental evidence that base pairing, commonly occurring between nucleic bases in more complex supramolecular arrangements, may affect the reaction pathways associated with the alkylation of bases themselves. In pursuit of this aim, dilute aqueous solutions of Cytidine- (CMP) and Guanosine-Mono-Phosphate (GMP) as single reactants or in an equimolar mixture were treated with the electrophilic alkylating agent 1,2- Dodecyl-Epoxide (DE), which was preventively dispersed into micellar solutions prepared with the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). In the early stage of the reaction, CTAB micelles acted as micro-heterogeneous nanoreactors, but as the reaction progresses the systems evolved toward the formation of polydisperse aggregates, whose size and surface-charge properties were monitored as a function of reaction time. From mass spectrometry analyses, it was found that the deamination of cytosine, a side reaction related to the alkylation of the amino group of CMP, was reduced when both the complementary ribonucleotides were present in the same reaction mixture. The involvement of specific sites able to establish C:G interactions (possibly via H-bonding or p–p stacking) could explain the reduced reactivity occurring at the level of some of the nucleophilic centers responsible for molecular recognition.

The Maillard-reaction-induced lactosylation of the major whey proteins, alpha-lactalbumin (alpha-La) and beta-lactoglobulins (beta-Lg) A and B, occurring upon heating at 70, 80 and 90 degrees C for 1 to 5 h in the presence of lactose excess, was studied by HPLC coupled to electrospray ionization single and tandem mass spectrometry (HPLC-ESI-MS, MS/MS). The presence of significant amounts of mono and bi-lactosylated forms of the three proteins and their increase with heating temperature and time were assessed from MS data. Evidences for a concomitant, significant denaturation, involving partial tertiary structure unfolding, were also obtained in the case of beta-lactoglobulins. A subsequent ESI-MS and MS/MS investigation on the tryptic digests of heated protein solutions exhibiting high percentages of mono and bi-lactosylated forms provided information on lactosylation sites. In particular, the latter were identified both on tryptic and on aspecific peptides, whose unusual relevance (compared to similar studies) was found to be due mainly to heat-induced protein degradation, occurring before protein digestion with trypsin. Among lactosylation sites identified only on tryptic peptides, i.e., those reasonably related to intact protein lactosylation, two lysines residues were found for alpha-La, both located in accessible regions of its tertiary structure. In the case of beta-Lg, besides three sites common to variants A and B (leucine 1, lysines 70, and 75), lysine 69 was found to be lactosylated only in variant B. Its proximity to a critical region of beta-Lg tertiary structure suggests that the difference between the two variants could be ascribed to a different evolution of their conformation upon heating.

Direct analysis in real time-high resolution mass spectrometry (DART-HRMS) was applied to the detection of lipid species in the lipid extracts of farmed salmon samples collected from a local retailer and analyzed right after the purchase and after storage for 4 and 6 days under refrigerated conditions. The recognition of type and composition of lipids detected in DART-HRMS spectra was performed by using the relevant accurate m/z data (accuracy better than 5 ppm) as input for a search on the LipidMaps database. As a result, several fatty acids (FA), either saturated or mono-/poly-unsaturated, and triacylglycerols (TAG) were recognized in the three types of samples from the corresponding negative and positive ion DART-HRMS spectra, respectively. Following, spectral intensities were exploited to monitor the evolution of selected FA and TAG during the refrigeration of salmon meat. In particular, after 4 days of refrigeration, a statistically significant increase was recorded for FA with side chain compositions 18:2, 18:1, 20:5, and 22:6 despite a significant decrease found for TAG with overall side chain compositions 50:4, 52:5, 52:4, and 52:3 after the same time. These evolutions were consistent with a general model already proposed for the effect of low temperature treatments on seafood, implying the action of endogenous lipases, with consequent increase of the free FA amount and decrease in glycerophospholipids and triglycerides contents. The described results indicate DART-HRMS as a promising MS-based rapid tool for the assessment of fish, or other seafood, freshness.

This study is aimed at making clear the relationship between oxidative stress of the phospholipid bilayer and membrane fluidity. Di-(hydroperoxylinoleoyl)-phosphatidylcholine (diHpLPC) was used as a highly hydroperoxidized and unsaturated phospholipid species in order to investigate the issue. Hydrophylic Interaction Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (HILIC-ESI-MS) and NMR spectroscopy were employed to define the structure of the peroxidized phospholipid as 1-(9-hydroperoxy-10c,12t)octadecadienoyl-2-(9t,11c-13-hydroperoxy)octadecadienoyl-sn-glycero-3-phosphorylcholine. This phospholipid's ability to form vesicular structures was confirmed by Sepharose 4B gel filtration and Dynamic Light Scattering (DLS) of its aqueous suspensions. Fatty acid misalignment and fluidity gradient were studied in the bilayer of both supported planar bilayers (SPB) and multilamellar vesicles (MLV) made of different DLPC/diHpLPC mixtures by means of spin labelling-EPR spectroscopy of either n-DSPC or 3-doxylcholestane spin labels embedded in the membranes. It was found that diHpLPC increases both fatty acid misalignment and rigidification with increasing molar ratio in spite of increasing unsaturation of the fatty acid core. Basing on our observations, the observed ability of pure diHpLPC to form rigid and disordered SPB and MLV bilayers is proposed to be dependent on the cross bridging of oxidized linoleoyl chains by mutual hydrogen bonding of hydroperoxyl groups. However, the contribution to the observed overall rigidification of the model membranes by trans double bonds in the peroxidized chains should not be neglected, as a second membrane fluidity effector also arising from lipid peroxidation.

An unprecedented characterization of free fatty acids (FFA) in the lipid extracts of fresh or thermally treated mussels of sp. Mytilus galloprovincialis, including up to 128 saturated, mono- or poly-unsaturated and 63 oxidized (i.e., modified by hydroxylic, carbonylic and/or epoxylic groups) compounds, was achieved using reverse phase chromatography coupled to electrospray ionization-Fourier transform single and tandem mass spectrometry (RPC-ESI-FTMS,MS/MS). Subsequent Principal Components Analysis (PCA) evidenced several effects of thermal treatments on the mussel FFA profiles. In particular, death-inducing low temperature treatments (freezing at -16 °C or refrigeration at 4 °C for several days) induced a peculiar increase in the incidence of FFA, whereas the effect was absent in mussels undergoing death upon prolonged storage at room temperature (25 °C, 6 h) or fast cooking (100 °C, 5 min). Alive mussels, either fresh or resulting from short term (up to 48 h) refrigeration were actually indistinguishable by PCA, although subtle seasonal effects were observed.

A mixture of native and oxidized phospholipids (PLs), generated by the soybean lipoxygenase type V-catalyzed partial oxidation of a lipid extract obtained from human platelets, was analyzed by HydrophilicInteraction Liquid Chromatography-ElectroSpray Ionization-Tandem Mass Spectrometry (HILIC–ESI-MS/MS). The complexity of the resulting mixture was remarkable, considering that the starting lipid extract, containing (as demonstrated in a previous study) about 130 native PLs, was enriched with enzy-matically generated hydroperoxylated derivatives and chemically generated hydroxylated forms of PLsbearing polyunsaturated side chains. Nonetheless, the described analytical approach proved to be verypowerful; indeed, focusing on phosphatidylcolines (PCs), the most abundant PL class in human platelets,about fifty different native/oxidized species could be identified in a single HILIC–ESI-MS/MS run. Low-energy collision induced dissociation tandem MS (CID-MS/MS) experiments on chromatographicallyseparated species showed single neutral losses of H2O2 and H2O to be typical fragmentation pathways of hydroperoxylated PCs, whereas a single H2O loss was observed for hydroxylated ones. Moreover, diag-nostic losses of n-hexanal or n-pentanol were exploited to recognize PCs hydroperoxylated on the last butfive carbon atom of a -6 polyunsaturated side chain. Despite the low resolution of the 3D ion trap mass analyzer used, the described HILIC–ESI-MS/MS approach appears very promising for the identification ofoxidized lipids in oxidatively stressed complex biological systems.

A method based on capillary liquid chromatography combined with electrospray ionization-tandem mass spectrometry (CapLC–ESI-MS–MS) for the detection and identification of casein deriving peptides in fined white wine is described. This is the first step towards the development of a liquid chromatography mass spectrometric method for the detection/identification of markers of potentially allergenic milk proteins used as wine fining agents. The method demonstrated to be capable of detecting some peptides arising from and casein (with the relative aminoacidic sequences elucidated) in extracts of white wine fined with casein at 100 and 1000g/mL. This MS based approach appears to be a useful tool for screening purposes as well as a confirmatory tool for the unequivocal identification of caseins in ELISA positive samples.

The identification of two unsaturated N-acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Along the confirmation of the signaling molecule already described in the literature, i.e. (Z)-N-tetradec-7-enoyl-homoserine lactone (C14:1-HSL), we have discovered the occurrence, at low yet significant levels, of another monounsaturated compound, C12:1-HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion to bromohydrins. Collision induced dissociation (CID) spectra were then collected on the LTQ mass analyser. A careful comparison of tandem MS spectra of monounsaturated (i.e., C12:1-HSL and C14:1-HSL) and saturated AHLs (i.e. C12-HSL and C14-HSL) led to emphasise two series of product ions, exhibiting 14 Da-spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e., CH2=CH(CH2)nH). In particular, the series located at the higher end of the explored m/z range (> 200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain.

Peroxidation catalysed by Soybean Lypoxigenase was performed on tetralinoleyl-cardiolipin with the aim of generating selectively oxidized products, to be used subsequently as standards for studies on cardiolipin oxidation. The reaction products were characterized by LC-ESI-MS and MS/MS, and the process was found to link a hydroperoxylic group on one or more linoleic chains of cardiolipin, up to a total of four groups per molecule. Interestingly, the incidence of other oxidized products, like those arising from multiple hydroxylation or mixed hydroxylation-hydroperoxydation, previously observed after the chemical oxidation of the same cardiolipin, was found to be negligible. Moreover, evidences for the presence of the hydroperoxylic group(s) almost exclusively on carbon 13 of the linoleic chain(s) were obtained by MS/MS measurements. The enzymatic approach, integrated with a preparative separation step, which could be developed by adapting the chromatographic conditions adopted in the present work for analytical purposes, represents a promising strategy for the synthesis of highly specific monoor multi-peroxidated derivatives of cardiolipins.

The phospholipidome of blood microparticles (MPs) obtained from platelet-rich plasma of healthy individuals was characterized by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The HILIC separation, performed on a silica stationary phase using an acetonitrile/methanol gradient, enabled the separation of several phospholipids (PL) classes, viz., phosphatidyl-cholines (PCs), -ethanolamines (PEs), -serines (PSs), -inositoles (PIs), sphyngomielins (SMs), and lyso forms of PCs and PEs. Structural characterization of species belonging to each class was performed by MS/MS measurements, in either positive or negative ion mode. The set of 131 phospholipids (including regioisomers) here identified represents the most comprehensive phospholipidomic characterization reported for human MPs. Although the phospholipidome composition of MPs and platelets, collected from the same donors, was found to be qualitatively the same, quantitative differences were evidenced for lyso-PCs, which appear to be significantly more abundant in MPs.

The pH-related characteristics of 4-thiothymidine and its stability during prolonged exposure, at room temperature, to a neon lamp emitting in the 400–700 nm wavelength range were investigated by different spectroscopic techniques (UV-Vis, FTIR-ATR, 1H-NMR) and by ElectroSpray Ionization Mass Spectrometry (ESI-MS). The evaluation of the nucleoside photostability was performed as a control, with the perspective of studying its reactivity under the same conditions but in the presence of visible lightabsorbing photosensitizers, able to generate reactive oxygen species. The comparison between UV-Vis spectra recorded at different pH values in the 7–12 range suggested the presence of an equilibrium related to the deprotonation of the N3–H group of 4-thiothymidine, with a pKa estimated to be close to 9. Some effects of the deprotonation occurring at alkaline pH were observed also in FTIR-ATR spectra, the main feature being the appearance of a band related to C]N stretching, interpreted with the assumption of a partial double bond character by C2–N3, N3–C4 and C2–O bonds, as a consequence of negative charge delocalization on the pyrimidine ring. As for photostability, UV-Vis, FTIR-ATR and NMR measurements suggested the generation of thymidine as a by-product but only after a prolonged (48 hours) irradiation time, whereas no significant alteration occurred in a shorter time range (1–2 hours), i.e. the one that will be considered in future studies involving the presence of photosensitizers. The nucleoside stability up to 2 hours of irradiation was confirmed by ESI-MS analyses; furthermore, on the other hand, the latter indicated the presence of three additional by-products, besides thymidine, after 48 hours of irradiation. In particular, an hydroxylated form of 4-thiothymidine and two dimeric species, characterized by S–S and S–O covalent bridges between two 4-thiothymidine and a 4-thiothymidine and a thymidine molecule, respectively, were detected.

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the alpha -amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSn, n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides. (C) 2015 Elsevier B.V. All rights reserved.

Reliable methods are needed for detection of allergenic milk proteins in complex food matrixes. The feasibility of an LC/high-resolution MS method for the analysis of milk proteins in a thermally processed model food (incurred cookies) and in white wine spiked, respectively, with milk powder and caseinate is described. Detection of milk proteins was based on the identification of unique peptides in the tryptic digests of cookie/wine extracts using an RP-HPLC separation coupled to an Exactive™ nonhybrid mass spectrometer using Orbitrap technology. The extremely high mass accuracy and resolution provided by the Orbitrap analyzer allowed a fast preliminary identification of four previously proposed peptide markers of caseins using only accurate values of the m/z of their ions. No interference was observed, despite the complexity of the analyzed matrixes. Moreover, the availability of a high- energy, collisionally activated dissociation cell integrated in the mass spectrometer enabled acquisition of peptide MS/MS-like spectra through post-source fragmentation. Confirmation of peptide marker identity could then be achieved by a comparison between experimental and predicted product ions. The described method shows the great potential of Orbitrap MS as a reliable technique in the field of protein allergen detection once the peptide markers are identified.

Oleuropein (Ole) has been claimed to mitigate cisplatin (CP) induced acute injury in kidney and liver of mice. In-vitro reactivity of hydrated CP species with Ole, and Ole metabolite hydroxytyrosol (HT) is of great interest as the preliminary step for gathering in-vivo information on the possible physiological role of the Ole/HT-cis-diammineplatinum (II) (Ole/HT-cis-DAP) conjugate.

Low temperature treatments commonly applied to seafood products have been shown to influence their phospholipid (PL) profile through enzymatic hydrolysis. In the present study, the generation of lysophospholipids (LPL) resulting from this process was systematically investigated for selected, commercially relevant seafood products, namely oysters, clams, octopuses, and shrimps. These products were subjected to thermal treatments like refrigeration or freezing after being purchased as fresh, defrozen, or frozen products depending on the case. The coupling between hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization with high resolution/accuracy Fourier transform mass spectrometry (ESI-FTMS) was exploited to evaluate the PL profile of the cited products, especially the incidence of LPL related to the two main PL classes of seafood products-phosphatidylcholines (PC) and phosphatidylethanolamines (PE)-in the lipid extracts. The lyso forms of PE (LPE) were found to be generally more sensitive than those of PC (LPC) to thermal treatments, usually exhibiting a significant increase upon prolonged refrigeration at 4 °C in all types of investigated products except European flat oysters. Moreover, the distinction between fresh and frozen or defrozen products could be achieved in the case of octopuses and shrimps, respectively.