Comparative Volatilomic Profile of Three Finger Lime (Citrus australasica) Cultivars Based on Chemometrics Analysis of HS-SPME/GC-MS Data.

Finger lime is receiving growing attention as an ingredient of gastronomic preparations of haute cuisine for its delicious flavor and fragrance and for its appealing aspect. Volatile compounds play a crucial role in determining the organoleptic characteristics of the fruit and its pleasantness for consumers. The aim of the present study was to investigate the volatile profiles by headspace solid phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) in the peel and, for the first time, in the pulp of three Australian finger lime cultivars grown in Sicily (southern Italy): Pink Pearl, Sanguinea, and Faustrime, allowing to overall identify 84 volatile organic compounds (VOCs). The analytical data showed that the three cultivars were characterized by distinct volatile chemotypes: limonene/sabinene/bicyclogermacrene in the Pink Pearl, limonene/γ-terpinene/bicyclogermacrene in the Sanguinea, and limonene/ß-phellandrene/γ-terpinene in the Faustrime. Moreover, some volatiles, found exclusively in one cultivar, could be considered potential markers of the individual cultivar. PCA allowed us to clearly discriminate the samples into three clusters: the first related to the Sanguinea peel, the second to the Faustrime peel, and a third group associated with the Pink Pearl peel along with the pulp of the three cultivars. Accordingly, the VOCs that mostly contributed to the differentiation of the three finger lime cultivars were also identified. Among them, D-limonene, sabinene γ-terpinene, α-pinene, α-phellandrene, ß-myrcene, p-cymene, linalool, δ-elemene, ledene, bicyclogermacrene ß-citronellol, α-bergamotene, α-caryophillene, and ß-bisabolene, have been previously reported to exhibit important biological activities, suggesting that these cultivars, in addition to possessing unique volatile profiles, can show promise for several applications in pharmaceutical and food industry, namely for development of functional foods.

Use of solid-phase microextraction coupled to gas chromatography-mass spectrometry for determination of urinary volatile organic compounds in autistic children compared with healthy controls.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which have a severe life-long effect on behavior and social functioning, and which are associated with metabolic abnormalities. Their diagnosis is on the basis of behavioral and developmental signs usually detected before three years of age, and there is no reliable biological marker. The objective of this study was to establish the volatile urinary metabolomic profiles of 24 autistic children and 21 healthy children (control group) to investigate volatile organic compounds (VOCs) as potential biomarkers for ASDs. Solid-phase microextraction (SPME) using DVB/CAR/PDMS sorbent coupled with gas chromatography-mass spectrometry was used to obtain the metabolomic information patterns. Urine samples were analyzed under both acid and alkaline pH, to profile a range of urinary components with different physicochemical properties. Multivariate statistics techniques were applied to bioanalytical data to visualize clusters of cases and to detect the VOCs able to differentiate autistic patients from healthy children. In particular, orthogonal projections to latent structures discriminant analysis (OPLS-DA) achieved very good separation between autistic and control groups under both acidic and alkaline pH, identifying discriminating metabolites. Among these, 3-methyl-cyclopentanone, 3-methyl-butanal, 2-methyl-butanal, and hexane under acid conditions, and 2-methyl-pyrazine, 2,3-dimethyl-pyrazine, and isoxazolo under alkaline pH had statistically higher levels in urine samples from autistic children than from the control group. Further investigation with a higher number of patients should be performed to outline the metabolic origins of these variables, define a possible association with ASDs, and verify the usefulness of these variables for early-stage diagnosis.

Influence of Plant-Based Biostimulant (BORTAN) on Qualitative and Aromatic Traits of Rocket Salad (Diplotaxis tenuifolia L.).

In this study, the influence of a new plant-based biostimulant (Bortan) on physiological and aromatic traits of rocket (Diplotaxis tenuifolia L. var. Pamela) was monitored by evaluating physico-chemical parameters (fresh and dry weight, leaf color and chlorophyll content) and biochemical traits (total phenolic compound (TP), total flavonoids (TF), ascorbic acid (AA) and antioxidant activity (AOX). Volatile profiles were also analyzed by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry, allowing the detection of 32 volatiles belonging to 5 chemical classes. Compared to the control, Bortan application enhanced leaf pigment content, including chlorophyll a, b and carotenoids (+10%, +16% and +28%, respectively) and increased TP (+34%), TF (+26%), AA (+19%) amonts and AOX value (+16%). Principal component analysis revealed a significant discrimination between the two samples. Specifically, treated samples were mainly associated with "green-leaf" volatiles, namely hexanal and 2-hexenal, 3-hexenal and 1-penten-3-one, while control rocket was directly correlated with several alcohols and to all isothiocyanates, associated with the sulfur-like odor of rocket. These findings can add further support, both for farmers and the agro-food industry, in choosing PBs as a new and sustainable practice in complementing enhanced yields with premium-quality produce. To confirm these preliminary data, further experiments are needed by enlarging the sample size, testing different concentrations of Bortan and/or using other food crops.

Agronomic, Physicochemical, Aromatic and Sensory Characterization of Four Sweet Cherry Accessions of the Campania Region.

Sweet cherries (Prunus avium L.) are greatly appreciated fruits worldwide due to their taste, color, nutritional value, and beneficial health effects. The characterization of autochthonous germplasm allows to identify genotypes that possess superior characteristics compared to standard cultivars. In this work, four accessions of sweet cherry from the Campania region (Limoncella, Mulegnana Riccia, Mulegnana Nera and Montenero) were investigated for their morpho-physiological, qualitative, aromatic, and sensorial traits in comparison with two standard cultivars (Ferrovia and Lapins). A high variability in the pomological traits resulted among the samples. Montenero showed comparable fruit weight and titratable acidity to Ferrovia and Lapins, respectively. The highest total soluble solid content was detected in Mulegnana Riccia. A considerable variability in the skin and pulp color of the cherries was observed, varying from yellow-red in Limoncella to a dark red color in Montenero. Mulegnana Nera showed the highest content of polyphenols, flavonoids, anthocyanins, and ascorbic acid compared to the standard cultivars. Volatile organic compounds profile analysis identified 34 volatile compounds, 12 of which were observed at different concentrations in all the sweet cherry genotypes while the others were genotype-dependent. Conservation and cultivation of autochthonous accessions with suitable nutritional and morpho-physiologic characteristics promotes our agrobiodiversity knowledge and allows to better plan future breeding programs.

Rapid and Non-Destructive Techniques for the Discrimination of Ripening Stages in Candonga Strawberries.

Electronic nose (e-nose), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and image analysis (IA) were used to discriminate the ripening stage (half-red or red) of strawberries (cv Sabrosa, commercially named Candonga), harvested at three different times (H1, H2 and H3). Principal component analysis (PCA) performed on the e-nose, ATR-FTIR and IA data allowed us to clearly discriminate samples based on the ripening stage, as in the score space they clustered in distinct regions of the plot. Moreover, a correlation analysis between the e-nose sensor and 57 volatile organic compounds (VOCs), which were overall detected in all the investigated fruit samples by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME/GC-MS), allowed us to distinguish half-red and red strawberries, as the e-nose sensors gave distinct responses to samples with different flavours. Three suitable broad bands were individuated by PCA in the ATR-FTIR spectra to discriminate half-red and red samples: the band centred at 3295 cm-1 is generated by compounds that decline, whereas those at 1717 cm-1 and at 1026 cm-1 stem from compounds that accumulate during ripening. Among the chemical parameters (titratable acidity, total phenols, antioxidant activity and total soluble solid) assayed in this study, only titratable acidity was somehow correlated to ATR-FTIR and IA patterns. Thus, ATR-FTIR spectroscopy and IA might be exploited to rapidly assess titratable acidity, which is an objective indicator of the ripening stage.

Biochemical Characterization of Some Varieties of Apricot Present in the Vesuvius Area, Southern Italy.

The witnesses of the millenary history of Campania felix in southern Italy highlighted that several fruit and vegetables cultivated in such territory could potentially be a treasure trove of important health elements. Our work evaluated the content of ß-carotene, ascorbic acid, and total phenolics and the antioxidant activity of ten typical varieties of apricots cultivated in the Vesuvius area in the Campania region. The total polyphenols varied between 10.24 and 34.04 mg/100 g of a fresh sample. The amount of ascorbic acid also varied greatly, ranging from 2.65 to 10.65 mg/100 g of a fresh product. B-Carotene reached values up to 0.522 mg/100 g of the fresh sample. The correlation analysis performed, accounting for these parameters, showed that the antioxidant activity, calculated by 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) and azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) tests, was influenced mainly by the content of total polyphenols, with ρ = -0.762 and ρ = 0.875 when we considered DPPH and ABTS tests, respectively, slightly less by the content of ascorbic acid, and not by ß-carotene. The dendrogram clustered eight varieties into two main groups; on the other hand, two varieties ("Vitillo" and "Preveta bella") seemed hierarchically distant. The gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOCs), herein performed for the first time, demonstrated the influence of the varieties on the VOC profiles, both from a qualitative and semiquantitative perspective, discriminating the varieties in different clusters, each of which was characterized by specific notes. α-Terpinolene was the only terpene identified by GC-MS that appeared to affect the antioxidant activity.

New Mater-Bi, Biodegradable Mulching Film for Strawberry (Fragaria × Ananassa Duch.): Effects on Film Duration, Crop Yields, Qualitative, and Nutraceutical Traits of Fruits.

In the main strawberry areas of Southern Italy, cultivation is carried out by transplanting plants on raised beds (30-40 cm from ground level), mulched with black polyethylene (PE). This technique has becoming increasingly expensive due to the growing prices of plastic mulches, the cost to remove them at the end of crop cycle, and the difficulty to dispose of black, dirty plastic films. The main objective of this research was the replacement of PE mulch with a new biodegradable mulching film Mater-Bi®-based (Novamont), characterized by an increased permanence in the field designed for long crop life. In 2021, two Mater-Bi-based, black, 18 µm thick mulching films were tested under tunnel: N5 as innovative film and N18 as commercial standard film. Black PE film, 50 µm thick was the control. Strawberry cultivars 'Sabrina' and 'Elide' were cropped on the three mulching films according to a split plot design with four replications. Harvests lasted from March to June 2021. Cvs Sabrina and Elide yielded around 40 t ha-1, while the mean effect of mulching films did not point out differences between the biodegradable mulches and PE. In 4 out of 12 harvests we analyzed samples of fruits to assess the influence of mulches on the contents of °Brix, polyphenols, antioxidant activity, ascorbic acid, flavonoids, and anthocyanins. On average, °Brix was clearly improved in fruits on PE compared to biodegradable films, while all the other qualitative traits resulted in being more dependent on the cultivars and times of sampling effects. Overall, biodegradable mulches are a viable alternative to PE mulch, and the innovative N5 film appeared promising for the enhancement of durability of soil coverage in a long-lasting cycle.

Proteomic profiling of human melanoma metastatic cell line secretomes.

During the last few years, the incidence and mortality of human melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated with cancer progression with poor prognosis and survival. These processes are controlled by dynamic interactions between tumor melanocytes and neighboring stromal cells, whose deregulation leads to the acquisition of cell proliferation capabilities and invasiveness. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. To address the issue of the proteins secreted during cancer progression, the proteomic profiling of secretomes of cancer cell lines from different melanoma metastases of the same patient (PE-MEL-41, PE-MEL-47, and PE-MEL-43) was performed by applying a shotgun LC-MS/MS-based approach. The results provide a list of candidate proteins associated with the metastatic potential of PE-MEL melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e., SPARC, osteopontin). In addition, the extracellular matrix protein 1 that stimulates proliferation and angiogenesis of endothelial cells as well as the fibronectin, involved in cell adhesion and motility, were identified. The present work provides the basis to clarify the complex extracellular protein networks implicated in human melanoma cell invasion, migration, and motility.

Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7.

The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

Study of the fragmentation pattern of ketamine-heptafluorobutyramide by gas chromatography/electron ionization mass spectrometry.

Ketamine is an anaesthetic compound used in human and veterinary medicine with hallucinogen properties that have resulted in its increased illicit use by teenagers at rave parties. Although several gas chromatography/mass spectrometry (GC/MS) methods have been reported for the quantification of the drug both in urine and in hair, its electron ionization (EI) fragmentation after derivatization with different reagents has been not yet fully investigated. The present work reports the study of the fragmentation of ketamine, derivatized with heptafluorobutyric anhydride (HFBA-Ket), using gas chromatography/electron ionization mass spectrometry (GC/EI-MS). The complete characterization of the fragmentation pattern represented an intriguing exercise and required tandem mass spectrometry (MS(n)) experiments, high-resolution accurate mass measurements and the use of deuterated d(4)-ketamine to corroborate the proposed structures and to characterize the fragment ions carrying the unchanged aromatic moiety. Extensive fragmentation was observed, mainly located at the cyclohexanone ring followed by rearrangement of the fragment ions, as confirmed by the mass spectra obtained from the deuterated molecule. The GC/EI-MS analysis of HFBA-Ket will represent a useful tool in forensic science since high-throughput analyses are enabled, preserving both the GC stationary phase and the cleanliness of the mass spectrometer ion optics.

Biochemical composition and antioxidant activity of three extra virgin olive oils from the Irpinia Province, Southern Italy.

Extra virgin olive oil (EVOO), appraised for its healthy properties, represents an important element for the economy of several countries of the Mediterranean area, including Italy. Our study aimed to evaluate some biochemical characteristics (polyphenols and volatile compounds) as well as the antioxidant activity of three EVOOs obtained from the varieties Ravece, Ogliarola, and Ruvea antica, grown in the same field of an Irpinian village, Montella, in the Campania region, Southern Italy. Extra virgin olive oil Ruvea antica contained the greatest amount of total polyphenols and showed the highest antioxidant activity. Principal component analysis of the aromatic profiles indicated that the three EVOOs could be easily discriminated according to the cultivar. 1-Hexanol, 2-hexen-1-ol, 3-pentanone, representing the most abundant volatiles of the EVOO Ruvea antica, and 2-hexenal, which resulted the main component in EVOOs Ogliarola and Ravece, could be considered as markers to discriminate these three EVOOs, according to the ReliefF feature selection algorithm.

Antibacterial Activity of Three Extra Virgin Olive Oils of the Campania Region, Southern Italy, Related to Their Polyphenol Content and Composition.

Production of extra virgin olive oil (EVOO) represents an important element for the economy of Southern Italy. Therefore, EVOO is recognized as a food with noticeable biological effects. Our study aimed to evaluate the antimicrobial activity exhibited by the polyphenolic extracts of EVOOs, obtained from three varieties of Olea europea L. (Ruvea antica, Ravece, and Ogliarola) cultivated in the village of Montella, Avellino, Southern Italy. The study evaluated the inhibiting effect of the extracts against some Gram-positive and Gram-negative bacteria. Statistical analysis, used to relate values of antimicrobial activity to total polyphenols and phenolic composition, revealed a different behavior among the three EVOO polyphenol extracts. The method applied could be useful to predict the influence of singular metabolites on the antimicrobial activity.

In vitro intestinal epithelium responses to titanium dioxide nanoparticles.

Titanium dioxide (TiO2) is enclosed in many consumer products including pharmaceuticals, cosmetics, and foods. TiO2 (E171) is daily ingested as mixed nano- and submicron-sized particles since it is approved as a white colorant in Europe in a wide variety of food products, Noteworthy, the relevant risk assessment has never been satisfactorily concluded and growing alarms for human hazards deriving from TiO2 exposure are incrementally reported. The objective of the present study was to establish conceivable mechanisms by which nano-sized TiO2 particles affect physiological function of the intestinal epithelium layer. The well-established Caco-2 cell line differentiated for 21 days on permeable supports was used as a predictive model of the human intestinal mucosa to identify the biological response triggered by TiO2 particles. Exposure to 42 µg/mL TiO2 nanoparticles disrupted the tight junctions-permeability barrier with a prompt effect detectable after 4 h incubation time and wide effects on barrier integrity at 24 h. Transport and ultrastructural localization of TiO2 nanoparticles were determined by ICP-OES, TEM and ESI/EELS analysis, respectively. Nano-sized particles were efficiently internalized and preferentially entrapped by Caco-2 monolayers. Storage of TiO2 nanoparticles inside the cells affected enterocytes viability and triggered the production of pro-inflammatory cytokines, including TNF-α and IL-8. Taken together these data indicate that nano-sized TiO2 particles exert detrimental effects on the intestinal epithelium layer.

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS.

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.

Exposure to sub-10nm particles emitted from a biodiesel-fueled diesel engine: In vitro toxicity and inflammatory potential.

OBJECTIVES: The inflammatory effects of organic sub-10nm particles generated and emitted from a diesel engine fueled with a biodiesel and a commercial diesel oil are analyzed in this paper. Diesel combustion is the major sources of ultrafine particles (UFP) in the environment, particularly in urbanized areas. In the last years, there is an increasing use of biomass-derived fuels because they are a renewable source of energy that may mitigate climate change through the reduction of net CO2 with respect to conventional fossil fuels. Although there is a general agreement on biofuels ability to reduce conventional pollutants, new and potentially harmful pollutants can be formed during biofuel combustion. In particular, the emission of sub-10nm particles is strongly increased with respect to that of larger soot particles. METHODS: Organic sub-10nm particles are separated from larger sizes particulate matter by collection in water suspension for toxicological and inflammatory tests. After exposure to sub-10nm particles, the effects on proliferation, apoptosis and secretion of cytokines, chemokines and growth factors networks production is analyzed in immortalized non-tumorigenic human dermal keratinocyte cell line (HaCaT) and human alveolar epithelial-like cells (A549). RESULTS AND CONCLUSION: Nanoparticles exert different cytotoxic effects in the two cell lines, suggesting that the dermal way of exposure is more sensitive than the inhalant way. These differences are most evident in the secretion of pro-inflammatory, angiogenic and proliferative cytokines and chemokines whose expression is more finely modulated in HaCaT cells compared to A-549 cells. Considering the size of these particles, it is important to promote the culture of prevention also for the dermal way in particularly exposed workers.

An integrated approach based on multiplexed protein array and iTRAQ labeling for in-depth identification of pathways associated to IVF outcome.

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.

Purification and enzymatic properties of a peroxidase from leaves of Phytolacca dioica L. (Ombú tree).

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)

A Bowman-Birk inhibitor with anti-elastase activity from Lathyrus sativus L. seeds.

Four Bowman-Birk inhibitors, named LSI-1/4, were isolated and purified from Lathyrus sativus L. seeds. The purification procedure consisted of two cation-exchange chromatography steps, followed by gel-filtration and RP-HPLC. Mass spectrometry analysis of LSI-1/4 inhibitors yielded relative molecular masses of 7914.41 for LSI-1, 6867.67 for LSI-2, 7341.24 for LSI-3 and 7460.01 for LSI-4. N-terminal sequences (up to 30 residues) of LSI-1/4 inhibitors were identical with the exception of sequence positions 21, 27 and 28 and highly similar to those of other Bowman-Birk inhibitors isolated from Leguminosae plants. Inhibitors LSI-1/4 were active towards trypsin and α-chymotrypsin, with IC(50) values for 12.6 nM of trypsin ranging from 4.9 to 24.3 nM. A lower activity was observed against bovine α-chymotrypsin (IC(50) values ranging from 0.5 to 3.4 µM for 15.0 nM of α-chymotrypsin). Peptide mapping of the LSI-1 sequence showed the presence of an Ala residue in the second reactive site, thus explaining the low anti-chymotrypsin activity of this inhibitor. In addition, LSI-1 was endowed with anti-elastase activity, being able to inhibit human leukocyte elastase.

Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B.

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.