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The evaluation of double bond positions in fatty acyl chains has always been of great concern given their significance in the chemical and biochemical role of lipids. Despite being the foremost technique for lipidomics, it is difficult in practice to obtain identification beyond the fatty acyl level by the sole high-resolution mass spectrometry. Paternò-Büchi reactions of fatty acids (FAs) with ketones have been successfully proposed for pinpointing double bonds in FAs in combination with the collision-induced fragmentation technique. In the present paper, an aza-Paternò-Büchi (aPB) reaction of lipids with 6-azauracil (6-AU) was proposed for the first time for the determination of carbon-carbon double bonds in fatty acyl chains using higher collisional dissociation in the negative ion mode. The method was optimized using free FA and phospholipid analytical standards and compared to the standard Paternò-Büchi reaction with acetone. The introduction of the 6-AU moiety allowed enhancing the ionization efficiency of the FA precursor and diagnostic product ions, thanks to the presence of ionizable sites on the derivatizing agent. Moreover, the aPB derivatization allowed the obtention of deprotonated ions of phosphatidylcholines, thanks to an intramolecular methyl transfer from the phosphocholine polar heads during ionization. The workflow was finally applied for pinpointing carbon-carbon double bonds in 77 polar lipids from an yeast (

Purpose: We previously identified novel thiazole derivatives able to reduce histone acetylation and histone acetyltransferase (HAT) activity in yeast. Among these compounds, 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) has been selected and used throughout this study.Experimental Design: The effect of CPTH6 on histone acetylation, cell viability and differentiation, cell-cycle distribution, and apoptosis in a panel of acute myeloid leukemia and solid tumor cell lines has been evaluated.Results: Here, we showed that CPTH6 leads to an inhibition of Gcn5 and pCAF HAT activity. Moreover, it inhibits H3/H4 histones and α-tubulin acetylation of a panel of leukemia cell lines. Concentration- and time-dependent inhibition of cell viability, paralleled by accumulation of cells in the G0/G1 phase and depletion from the S/G2M phases, was observed. The role of mitochondrial pathway on CPTH6-induced apoptosis was shown, being a decrease of mitochondrial membrane potential and the release of cytochrome c, from mitochondria to cytosol, induced by CPTH6. Also the involvement of Bcl-2 and Bcl-xL on CPTH6-induced apoptosis was found after overexpression of the two proteins in leukemia cells. Solid tumor cell lines from several origins were shown to be differently sensitive to CPTH6 treatment in terms of cell viability, and a correlation between the inhibitory efficacy on H3/H4 histones acetylation and cytotoxicity was found. Differentiating effect on leukemia and neuroblastoma cell lines was also induced by CPTH6.Conclusions: These results make CPTH6 a suitable tool for discovery of molecular targets of HAT and, potentially, for the development of new anticancer therapies, which warrants further investigations. Clin Cancer Res; 18(2); 475–86. ©2011 AACR.

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This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC–MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC–MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird’s-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic. Graphical Abstract

Blueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet.In the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content.V. floribundum native and fermented berries were extracted and analyzed by UHPLC-HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification.In total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds.Andean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

In recent years, there is increasing attention on the contaminants of emerging concern (CECs), which include plasticizers, flame retardants, industrial chemicals, pharmaceuticals, and personal care products, since they have been detected even far away from pollution sources. The polar regions are not exempt from the presence of anthropogenic contaminants, and they are employed as a model for understanding the pollutant fate and impact. During the 2021 spring campaign, sixteen surface snow samples were collected close to the research station of Ny-Ålesund located on the Spitsbergen Island of the Norwegian Svalbard Archipelago. The samples were extracted by solid-phase extraction and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) following an untargeted approach. Compound tentative identification was obtained with the aid of the software Compound Discoverer, using both mass spectral database search and manual validation. Among the 114 compounds identified with a high confidence level in the snow samples,80 have some commercial or industrial use (drugs, plasticizers, fragrances, etc.), therefore they could be of anthropogenic origin. Nonetheless, a clear contamination trend did not appear in the snow samples collected on eight different days during one month. The comparison with aerosol samples collected in the same area did not help identifying the source, either, since only a few compounds were in common, and they were mainly of natural origin. As such, the analysis of aerosol sample did not support possible long-range transport, also considering that compounds were detected mostly in the coarse fraction.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost‐effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n‐hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high‐resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78‐100%), intra‐ and interday relative standard deviations ( 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.

A highly stereoselective procedure for the synthesis of spiro-polycyclic oxindoles bearing five contiguous stereogenic centers including two tetrasubstituted carbons has been developed. Under sequential organocatalysis performed by a pyrrolidine-based organocatalyst and DBU, a highly atom-economical Michael–domino Michael/aldol reaction sequence was optimized, yielding variously functionalized spiro-decalin oxindoles with excellent stereoselectivity (>99:1 dr, up to 92% ee). peerReviewed

This study aims to obtain a valuable mixture of short-chain peptides from hempseed as a new ingredient for developing nutraceutical and functional foods useful for preventing metabolic syndrome that represents the major cause of death globally. A dedicated analytical platform based on a purification step by size exclusion chromatography or ultrafiltration membrane and high-resolution mass spectrometry was developed to isolate and comprehensively characterize short-chain peptides leading to the identification of more than 500 short-chain peptides. Our results indicated that the short-chain peptide mixture was about three times more active than the medium-chain peptide mixture and total hydrolysate with respect to measured inhibition of the angiotensin-converting enzyme. The short-chain peptide mixture was also two times more active as a dipeptidyl peptidase IV, and twofold more active on the cholesterol metabolism pathway through the modulation of low-density lipoprotein receptor.

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode. Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the "bioelectric well". Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses. The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from -0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Metaproteomic analysis of aquifer systems provides valuable information on the microbial populations, their influence on drinking water quality, and the effect on human health. In the present paper, an extraction and enrichment method by C18 extra-wide pore cartridge was developed, optimized, and applied for the first time to the metaproteomic characterization of dissolved organic matter in groundwater samples. In particular, three elution procedures were tested and compared on water spiked with a yeast protein extract to maximize the recovery of proteins from a complex matrix. The maximum protein recovery was obtained by the use of two sequential elution buffers, one employing a denaturing agent and the other one containing an acidified organic solvent. A comprehensive metaproteomic analysis of the dissolved organic matter of groundwater was then performed by nano-high performance liquid chromatography coupled to high-resolution mass spectrometry. A total of 239 proteins was identified; in agreement with the current knowledge on proteins in aquifer systems, most identified sequences derived from bacteria, protobacteria, and ciliates. The paper is the first metaproteomic study applied to groundwater samples with particular emphasis on the need for sample pretreatment to obtain comprehensive information on the proteome in dissolved organic matter.

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham.

This paper describes the process of design, analysis, and scoring with both subjective and objective criteria, of a set of possible haptic inceptors configurations to be used as primary flight control devices for a class of future tilt rotor vehicles. Several scoring criteria were adopted like ergonomics, cognitive compatibility, easiness of recovery in fault situations, and expected workload among several others. Each criterion was weighted to obtain a scoring matrix for a total of 18 different configurations, some "classic", some novel, leading to an overall ranking. The matrix was used to select the best candidate configurations to be tested using a human-in-the-loop simulator in the context of the EU Funded CleanSky2 SMARTeR Project. After a final review phase, the best solution will be adopted to actually fly the Leonardo NextGen Civil Tilt Rotor Technological Demonstrator in the context of the EU Funded CleanSky2 SAIS Project.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 μg mL−1). The untargeted characterization revealed that (−)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 μg mL−1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples. Four materials were prepared featuring a polymeric shell with tunable hydrophilicity, obtained by “grafting from” polymerization of glycidyl methacrylate with 0–8.3% of polyethylene glycol methacrylate (PEGMA), the latter used to modulate the hydrophilicity of the material surface. Finally, the materials were functionalized with iminodiacetic acid for Ti4+ ion immobilization. The materials were analyzed for their composition by a combination of CHN elemental analysis and thermogravimetric analysis, also hyphenated to gas chromatography and mass spectrometric detection. Surface characteristics were evaluated by water contact angle measurements, scanning electron microscopy and energy dispersive X-ray spectrometry. These materials were applied to the enrichment of phosphopeptides from yeast protein digests. Peptides were identified by proteomics techniques using nano-high performance liquid chromatography coupled to mass spectrometry and bioinformatics. Qualitatively the peptides identified by the four systems were comparable, with 1606–1693 phosphopeptide identifications and a selectivity of 47–54% for all materials. The physico-chemical features of the identified peptides were also the same for the four materials. In particular, the grand average of hydropathy index values indicated that the enriched phosphopeptides were hydrophilic (ca. 90%), and only some co-enriched non-phosphorylated peptides were hydrophobic (21–28%), regardless of the material used for enrichment. Peptides had a pI ≤ 7, which indicated a well-known bias for acidic peptides binding, attributed to the interaction with the metal center itself. The results indicated that the enrichment of phosphopeptides and the co-enrichment of non-phosphorylated peptides is mainly driven by interactions with Ti4+ and does not depend on the amount of PEGMA chains in the polymer shell.

This minireview is dedicated to the discussion of analytical methods based on magnetic solid-phase extraction for the investigation of different analyte classes in complex matrices. Magnetic solid-phase extraction represents one of the most exploited approaches for sample preparation, which benefits from the development of new materials and from the coupling with other purification and clean-up strategies. New materials are continuously described for the isolation and enrichment of a variety of compounds, from small molecules to biologic macromolecules. Such magnetic materials developed for magnetic solid-phase extraction are discussed in this minireview, spanning across different types of materials, from the more traditional magnetic nanoparticles functionalized with polymers, to molecularly imprinted polymers, but also graphene, carbon nanotubes, graphitized carbon black, metal organic frameworks, covalent organic frameworks, composite materials, biopolymers (polydopamine, chitosan), materials from wastes and natural products and the newly introduced knitting aromatic polymers. The magnetic solid-phase extraction methods are collected from the recent literature and organized in sections based on the target analyte classes, which include drugs, endocrine-disrupting compounds, pesticides, polycyclic aromatic hydrocarbons, metals, toxins, peptides, proteins, metabolites and a final chapter dedicated to applications to other common pollutants, contaminants and multiresidue methods. A selection of recent applications and variations of the traditional magnetic solid-phase extraction protocols is discussed for food, environmental and biologic matrices. Finally, the compliance of magnetic solid-phase extraction with the principles of green analytical chemistry is also briefly discussed, with recent examples, indicating the use of waste or sustainable materials, development of green material preparations and reduction of organic solvents as the main strategies for future development of environmentally friendly magnetic solid-phase extraction methods.

An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 μg kg−1. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only l -SeMet was found at level MQL (0.2 μg kg−1)-1.42 μg kg−1 in ten samples, while total Se was in the range of MDL-9.1 μg kg−1. Concentration of l -SeMet (5–6% of total Se) and total Se correlated very well to each other (R2 = 0.995).

This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analyzers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.

Soybeans (Glycine max) are an excellent source of dietary proteins and peptides with potential biological activities, such as antihypertensive, anti-cholesterol, and antioxidant activity, moreover, they could prevent cancer. Also, soy contains all the essential amino acids for nutrition, therefore, it represents an alternative to animal proteins. The goal of this paper was the comprehensive characterization of medium-sized and short peptides (two to four amino acids) obtained from simulated gastrointestinal digestion. Two different analytical approaches were employed for peptide characterization, namely a common peptidomic analysis for medium-sized peptides and a suspect screening analysis for short peptides, employing an inclusion list of exact m/z values of all possible amino acid combinations. Moreover, fractionation by preparative reversed-phase liquid chromatography was employed to simplify the starting protein hydrolysate. Six fractions were collected and tested for antioxidative activity by an innovative antioxidant assay on human gastric adenocarcinoma AGS cell lines. The two most active fractions (2 and 3) were then characterized by a peptidomic approach and database search, as well as by a suspect screening approach, in order to identify potential antioxidant amino acid sequences. Some of the peptides identified in these two fractions have been already reported in the literature for their antioxidant activity.

Prostate cancer, a leading cause of cancer-related deaths worldwide, principally occurs in over 50-year-old men. Nowadays there is urgency to discover biomarkers alternative to prostate-specific antigen, as it cannot discriminate patients with benign prostatic hyperplasia from clinically significant forms of prostatic cancer. In the present paper, 32 benign prostatic hyperplasia and 41 prostatic cancer urine samples were collected and analyzed. Polar and positively charged metabolites were therein investigated using an analytical platform comprising an up to 40-fold analyte enrichment step by graphitized carbon black solid-phase extraction, HILIC separation, and untargeted high-resolution mass spectrometry analysis. These classes of compounds are often neglected in common metabolomics experiments even though previous studies reported their significance in cancer biomarker discovery. The complex metabolomics big datasets, generated by the UHPLC-HRMS, were analyzed with the ROIMCR procedure, based on the selection of the MS regions of interest data and their analysis by the Multivariate Curve-Resolution Alternating Least Squares chemometrics method. This approach allowed the resolution and tentative identification of the metabolites differentially expressed by the two data sets. Among these, amino acids and carnitine derivatives were tentatively identified highlighting the importance of the proposed methodology for cancer biomarker research., The work was supported by the PRIN project Prot. 2017Y2PAB8, entitled “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”, provided by the Italian Ministry of Education, Universities and Research. Moreover, this work was also supported by Generalitat de Catalunya (Suport a les activitats de Grups de Recerca, 2017 SGR753).

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices.

Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.

Polyhydroxyalkanoates (PHAs) are well-known biodegradable plastics produced by various bacterial strains, whose major drawback is constituted by the high cost of their synthesis. Producing PHAs from mixed microbial cultures and employing organic wastes as a carbon source allows us to both reduce cost and valorize available renewable resources, such as food waste and sewage sludge. However, different types of pollutants, originally contained in organic matrices, could persist into the final product, thus compromising their safety. In this work, the exploitation of municipal wastes for PHA production is evaluated from the environmental and health safety aspect by determining the presence of polycyclic aromatic hydrocarbons (PAHs) in both commercial and waste-based PHA samples. Quantification of PAHs by gas chromatography-mass spectrometry on 24 PHA samples obtained in different conditions showed very low contamination levels, in the range of ppb to a few ppm. Moreover, the contaminant content seems to be dependent on the type of PHA stabilization and extraction, but independent from the type of feedstock. Commercial PHA derived from crops, selected for comparison, showed PAH content comparable to that detected in PHAs derived from organic fraction of municipal solid waste. Although there is no specific regulation on PAH maximum levels in PHAs, detected concentrations were consistently lower than threshold limit values set by regulation and guidelines for similar materials and/or applications. This suggests that the use of organic waste as substrate for PHA production is safe for both the human health and the environment.

Introduction: If the infectious focus is not identified, there is an associated higher mortality in Staphylococcus aureus bacteraemia (SAB) [1]. A retrospective observational study found patients with high-risk SAB who had a PET scan had a 67% reduction in mortality compared to those who did not [2]. We used electronic data to determine the range of infectious foci in SAB and the use of PET in our trust. Methods: From 1/1/13 to 31/12/18 all patients with SAB at St Thomas’ Hospital, London, were reviewed by the infectious diseases team and data collected prospectively using an Access database. A retrospective analysis of this database, electronic records, radiology and nuclear imaging was conducted. Results: 355 episodes of SAB affected 296 patients. 28 (8%) episodes were MRSA in 24 (8%) of patients. Infectious sources found included bone and joint (23%), IV access (15%), vascular infection (15%), no infective focus (13%), SSTI (12%) and endocarditis (9%). Imaging used to determine the focus of infection included MRI (18%), CT (26%) and PET scan (10%). In-hospital mortality was significantly lower in those who had a PET scan than in those who did not (1/31, 3% v. 50/265 patients (last episode), 19%, P=0.03). Conclusions: Mortality was lower in those who had PET at our trust. This is consistent with data published from other centres and requires further investigation. A prospective trial of PET in SAB is urgently needed. References: [1] PMID: 30179645 [2] PMID: 28336786

Among the organic contaminants that could pass from waste to polyhydroxyalkanoates (PHAs), there are the polycyclic aromatic hydrocarbons (PAHs). For this reason, we have developed a rapid analytical method for the determination of sixteen PAHs in PHAs. PAHs were extracted by n-hexane, after matrix dispersion and crumbling into sand; the extract was purified by solid phase extraction using florisil as adsorbent. Recoveries in the range of 89–101% were obtained for the deuterated analytes, except for the two with the lowest molecular weight. Trueness between 92% and 108% and within-laboratory precision (expressed as relative standard deviation) ≤18% were estimated for all the analytes. Gas chromatography/mass spectrometry was used for analyte determination. Method limits of quantification were suitable to assure that PAH presence in PHA biolpolymers is much below the limits set by European law for plastic materials. Indeed, analysis of two different PHA samples showed that contamination is limited to few compounds at non-concerning levels.

The paper describes the development of an enrichment method for the analysis of the endogenous phosphopeptides in saliva. The method was based on magnetic solid phase extraction by a magnetic graphitized carbon black-TiO2 composite material and was developed considering different saliva pre-treatments, namely C18 solid phase extraction for purification, direct dilution in loading buffer or acetonitrile precipitation. The method was based on a shotgun proteomics workflow and the enriched peptide mixture was analysed by nanoHPLC and high resolution tandem mass spectrometry. Acetonitrile precipitation provided the best results, with up to 165 endogenous phosphopeptides identified in saliva samples from healthy individuals. The physico-chemical features of the identified endogenous phosphopeptides indicated that such peptides were large, hydrophilic and basic.

Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity. Graphical abstract Workflow showing the entire peptidomic approach developed for identification of bioactive peptides in microalgae.

The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the “grafting from” approach by the activator regenerated by electron transfer–atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.

In this work, an untargeted approach based on ultra-high performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for the analysis of contaminants in surface water samples, possibly deriving from an ineffective removal by wastewater treatment plants or illicit release. First, solid-phase extraction (SPE) conditions for 30 target compounds were optimized for two different sorbents, namely the commercial Oasis HLB and graphitized carbon black (GCB), by Box-Behnken designs of experiments. Upon identification of optimal conditions, the two SPE materials yielded comparable extraction recoveries, ranging between 52-110% and 55-100% for Oasis HLB and GCB, respectively. Thereafter, river water samples collected in five locations near Rome (Italy) were extracted using both sorbents and then analyzed by UHPLC-HRMS/MS in both positive and negative electrospray (ESI) ion polarities. The retrieved raw data files were submitted to Compound Discoverer software and compound identification was tentatively assigned by matching their tandem mass spectra with the mzCloud mass spectral library and manual validation, resulting in a total of 241 identified compounds (211 and 40 in positive and negative ESI mode, respectively), mainly belonging to pharmaceuticals.