The rapidly increasing adoption of high-resolution accurate-mass methods in analytical laboratories has fueled demand for instruments that combine high performance and reliability with small size and greater ease-of-use. This paper presents the major design principles that are driving the evolution of the hybrid quadrupole-Orbitrap instrument architecture to enable a greater range of applications and users. These principles may be summarized as follows: better usage of physical space and better access for service by means of size reduction of pumping and ion optics; expanded use of technologies from electronics in ion-optical design; flexibility in performance via modularity of design of the hardware and software components; and, harmonization of interfaces with other instruments to facilitate sharing and transferability of analytical workflows. The design of a novel family of hybrid mass spectrometers is described in detail, and performance evaluation is carried out on a wide variety of samples for its three representatives: the Orbitrap Exploris 120, Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers.The new instrument family is shown to offer compelling potential not only for high-end proteomics and biopharmaceutical applications, but also for screening, trace, targeted and clinical analysis by liquid chromatography/mass spectrometry methods.

Exposure to malachite green (MG) may pose great health risks to humans, thus it is of prime importance to develop fast and robust methods to quantitatively screen the presence malachite green in environment. Herein application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 ug L-1 (S/N=3) in lake water samples and ~ 0.5 ug L-1 in ultrapure water under optimized experimental conditions. The signal intensity of MG showed good linearity over the concentration range of 10–5000 ug L-1. Measurement of practical water samples fortified with MG at 0.01 and 0.1 mg L-1 gave a good validation of the established calibration curve. The average recoveries of malachite green in lake water and Carassius carassius fish farm effluent water were 114.9% (6.6% RSD) and 85.4% (9.2% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples.

Metabolomics analysis generates vast arrays of data, necessitating comprehensive workflows involving expertise in analytics, biochemistry and bioinformatics, in order to provide coherent and high-quality data that enables discovery of robust and biologically significant metabolic findings. In this protocol article, we introduce NoTaMe, an analytical workflow for non-targeted metabolic profiling approaches utilizing liquid chromatography–mass spectrometry analysis. We provide an overview of lab protocols and statistical methods that we commonly practice for the analysis of nutritional metabolomics data. The paper is divided into three main sections: the first and second sections introducing the background and the study designs available for metabolomics research, and the third section describing in detail the steps of the main methods and protocols used to produce, preprocess and statistically analyze metabolomics data, and finally to identify and interpret the compounds that have emerged as interesting.

Background: Pittosporum tobira (Thunb.) Aiton is an aromatic medicinal plant widely cultivated in the world. However, the essential oils (EOs) from P. tobira flowers for anti-cancer potential is still not systematically studied. The present aim to elucidate the phytochemical composition of the EOs and to explore mechanism of anticancer action. Methods: The EOs were extracted and analyzed by headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS). Volatile components were identified according to Kovats retention index (KI) and NIST database. The anti-neoplasm mechanisms of the EOs was comprehensively investigated in lung carcinoma A549 and H460 cells. Results: A total of 47 secondary metabolites representing 94.18% of the EOs were successfully identified: monoterpenes and sesquiterpenes were the dominant terpenoids. The EOs exhibited antiproliferative activity on A549 and H460 lung carcinoma cells. Hoechst 33324 fluorescent staining indicated the typical characteristics of apoptosis and induced cycle phase arrest. AnnexinV/PI staining revealed that the number of apoptotic cells was increased. Furthermore, the EOs also induced the caspase-mediated mitochondrial apoptosis pathway. Conclusions: Findings suggest that the full-scale chemical composition and first characterization of anticancer activities of the EOs, it could be used for integrative natural anti-cancer agents in health care should be pay more attention.

Despite the increasing number of individuals affected by Alzheimer’s disease (AD) every year, no effective therapy has been developed to treat this neurodegenerative disease yet. The current methods for AD diagnosis are effective for clinical confirmation of the disease only when symptoms become apparent, years after molecular damage started within the patients’ brains. As higher expression of a conformationally altered p53 has been correlated with AD, we developed a mass spectrometry-based method for highly sensitive, specific, and reproducible quantification of a p53 conformational variant in plasma samples of patients with known clinical outcome. In particular, we tested the prognostic performance of an AD-specific 2D3A8-immunoselected p53 peptide (AZ 284™) in different sets of individuals progressing from both cognitively unimpaired (CU) and mild cognitive impairment (MCI) patients progressing to AD dementia. Our data showed that quantitative analysis of AZ 284™ is a reliable tool for predicting AD progression up to 6 years prior to dementia onset with AUC >90%. Taken together, these results support the implementation of p53 conformational variant quantification as an affordable and powerful diagnostic tool for early, non-invasive AD diagnosis.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The fragments were selected from patients with invasive ductal carcinoma of the breast, the most common and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers, useful for diagnostic and prognostic categorization of patients, in assessing post-surgical therapeutic regimes. The proteomic screening, by 2D-IPG and mass spectrometry, allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients, and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is generally supported by two concurrent pathways: the inhibition of apoptosis and the stimulation of cellular proliferation. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. To our knowledge this report on large-scales proteomics of breast cancer is currently a unique approach in the literature that offers the opportunity to evaluate the presence and recurrence of proteins to be used as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.

Corona viruses hijack human enzymes to assembly sugar coat on Spike glycoproteins. The mechanism that human antibodies may uncover the antigenic viral peptide epitopes hidden by sugar coat are unknown. In this study, we analyzed recombinant SARS-CoV-2 Spike protein secreted from BTI-Tn-5B1-4 cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired MS/MS spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of Spike proteins according to Cryo-EM and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. The results showed that all 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose type of N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the Spike receptor binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains included those located on Central Helix, between amino acids 967 and 1016 of SARS-CoV-1, and 985 to 1034 of SARS-CoV-2 Spike protein. As the majority of antibody paratopes bind to peptide portion with or without sugar modification, we propose a snake-catcher model that a minimal length of peptide is first clamped by a paratope, and the binding is either strengthened by sugars close to peptide, or not interfered by sugar modification.

The present work describes the optimization and validation of a highly selective and sensitive analytical method using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE LC-MS/MS) for the determination of some frequently prescribed pharmaceuticals in urban wastewater received and treated by Sharjah sewage treatment plant (STP). The extraction efficiency of different SPE cartridges was tested and the simultaneous extraction of pharmaceuticals was successfully accomplished using hydrophilic-lipophilic-balanced reversed phase Waters^® Oasis HLB cartridge (200 mg/ 6 mL) at pH 3. The analytes were separated on an Aquity BEH C₁₈ column (1.7 µm, 2.1 mm x 150 mm) using gradient elution and the mass spectrometric analysis were performed in multiple reactions monitoring (MRM) selecting two precursor ions to produce ion transition for each pharmaceutical using positive electrospray ionization (+ESI) mode. The correlation coefficient values in the linear calibration plot for each target compound exceeded 0.99 and the recovery percentages of the investigated pharmaceuticals were more than 84%. Limit of detection (LOD) varied between 0.1-1.5 ng/L and limit of quantification (LOQ) was 0.3-5 ng/L for all analytes. The precision of the method was calculated as the relative standard deviation (RSD%) of replicate measurements and was found to be in the ranges of 2.2% to 7.7% and 2.2% to 8.6% for inter and intra-day analysis, respectively. All of the obtained validation parameters satisfied the requirements and guidelines of analytical method validation.

Fabric phase sorptive extraction, an innovative integration of solid phase extraction and solid phase microextraction principles, has been combined with gas chromatography-mass spectrometry for the rapid extraction and determination of nineteen organochlorine pesticides in various fruit juices and water samples. FPSE consolidates the advanced features of sol-gel derived extraction sorbents with the rich surface chemistry of cellulose fabric substrate, which could directly extract target analytes from complex sample matrices and substantially simplifies the sample preparation operation. Important FPSE parameters including sorbent chemistry, extraction time, stirring speed, type and volume of back-extraction solvent and back-extraction time have been optimized. Calibration curves were obtained in the concentration range 0.1-500 ng/mL. Under the optimum conditions, the limits of detection were obtained in the range 0.007-0.032 ng/mL with satisfactory precision (RSD<6%). The relative recoveries obtained by spiking organochlorine pesticides in water and selected juice samples were in the range of 91.56–99.83%. The sorbent sol-gel poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) was applied for the extraction and preconcentration of organochlorine pesticides in aqueous and fruit juice samples prior to analysis with gas chromatography-mass spectrometry. The results demonstrated that the present method is simple, rapid, and precise for the determination of organochlorine pesticides in aqueous samples.

An understanding of the mechanism underlying fruit ripening is critical for fruit quality improvement. Although post-harvest ethylene application is known to enhance the onset of fruit ripening, exact mechanisms remain unclear. To characterize the fruit ripening process and mechanism, we investigated the effects of exposing kiwifruit cultivars ‘Hayward’ and ‘Gamrok’ to exogenous ethylene treatment post-harvest using comprehensive proteomic analyses. Comparative two-dimensional gel electrophoresis showed that most of the proteins aggregated in ethylene-treated samples compared to the control (non-treated). We observed that among all ethylene treatments, 95 proteins from ‘Hayward’ and 106 from ‘Gamrok’ were differentially expressed. Interestingly, among the elicited protein successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry, 50% from “Hayward’ and 60% from ‘Gamrok’ are associated with fruit ripening. Furthermore, 18% and 10% of proteins, respectively, are associated with defense response, whereas other major proteins are related to protein biosynthesis and photosynthesis/Calvin cycle. Interactions between identified proteins were demonstrated by bioinformatic analysis, providing insights into biological pathways and molecular functions in post-harvest kiwifruit ripening elicited by ethylene application. The present proteomic study in accordance with physiological analysis provides a quantitative evaluation of fruit ripening in response to exogenous ethylene in post-harvest kiwifruit.

Samples of sunflower Waste Cooking Oils (WCOs) subjected to several cycles of frying were treated with water under four different combinations of temperature and pH. The variation of the chemical composition of the oil samples was monitored through headspace Solid-Phase Microextraction (HS-SPME) coupled with gas-chromatographic technique (GC), ¹H NMR spectroscopy and ESI⁺ mass spectrometry measurements. The possibility to modify the chemical composition of the recycled vegetable oil by tuning the water treatment parameters was exploited for the designing of a mini-plant for the production of bio-lubricants. A full description of the proposed prototype is also reported.

Dynamic tribochemical processes for dicationic ionic liquid containing a germinal imidazolium cation head groups bridged by a poly(ethylene glycol) and bis(trifluoromethylsulfonyl)imide anion were studied using time-resolved Mechanically Stimulated Gas Emission Mass-Spectrometry (MSGE-MS). In comparison with similar monocationic imidazolium ionic liquids with short alkylic or long polyether side chains the dicationic ionic liquid had lower coefficient of friction on Ti6Al4V alloy and smoother behaviour. The analysis of volatile decomposition products suggested multiple tribochemical reactions in which both anionic and cationic moieties are involved. Tribochemical degradation of cations was mainly through detachment of the side and bridging chains from the imidazolium head groups. Absence of volatile products containing nitrogen implies that imidazole group remained unchanged. Hydrogen and water desorption were attributed to the reactions of hydrogen fluoride being a product of anion degradation with titanium and titanium oxide.

Rhizophora mangle L. is a well-known medicinal plant found in mangroves worldwide used to treatment diabetes. This study evaluated the chemical composition of the acetonic extract from Rhizophora mangle barks (AERM), by HPLC-PDA and FIA-ESI-IT-MS/MS and the effects on high-fat diet induced obesity in mice and its mechanism of action by gene expression of inflammatory markers (Pparg, Ppara, Srebf1, Cd36, Tnf, Ccl2, Lep, Il10, Il6, Fasn, 18s). High-fat diet fed mice during 12 weeks was used as model of obesity and associated alterations. The results were very satisfactory, the extract, rich in polyphenolic compounds, flavonoids and phenolic acids, displayed intense antioxidant activity in vitro (608 µmol Trolox/g), and showed excellent activity against non-alcoholic fatty liver disease (NAFLD) and reverse insulin resistance in a model of diet-induced obesity. We can registered a modulatory effect of AERM in liver PPAR-γ mRNA expression associated to an important inhibition of CD36 mRNA expression suggesting that AERM induces the down regulation of CD36 mRNA via PPAR-gamma inhibition. These results support the traditional knowledge about the use of R. mangle for the treatment of type 2 diabetes and reveal the potential of AERM for the treatment of NAFLD and management of obesity and comorbidities.

Glycosphingolipids (GSL) represent a highly heterogeneous class of lipids with many cellular functions, implicated in a wide spectrum of human diseases. Their isolation, detection, and comprehensive structural analysis is a challenging task due to the structural diversity of GSL molecules. In this work, GSL subclasses are isolated from human plasma using an optimized monophasic ethanol–water solvent system capable to recover a broad range of GSL species. Obtained deproteinized plasma is subsequently purified and concentrated by C18-based solid-phase extraction (SPE). The hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization linear ion trap tandem mass spectrometry (ESI-LIT-MS/MS) is used for GSL analysis in the human plasma extract. Our results provide an in-depth profiling and structural characterization of glycosphingolipid and some phospholipid subclasses identified in the human plasma based on their retention times and the interpretation of tandem mass spectra. The structural composition of particular lipid species is readily characterized based on the detailed interpretation of MS and MS/MS spectra and further confirmed by specific fragmentation behavior following predictable patterns, which yields to the unambiguous identification of 154 GSL species within 7 lipid subclasses and 77 phospholipids representing the highest number of GSL species ever reported in the human plasma. The developed HILIC-ESI-MS/MS method can be used for further clinical and biological research of GSL in the human blood or other biological samples.

Transplant of human cancer cells into zebrafish larvae has emerged as a useful methodology in cancer research. Zebrafish have very low husbandry costs, are amenable to large-scale drug screening, and are unmatched for optical clarity in live animal imaging. However, there is currently no consensus on the ideal methods for xenograft of human cancer cells into zebrafish. Here, we have examined the effects of transplant site and housing temperature on both zebrafish larvae and human cancer cells using survival analyses, metabolomic approaches, and in vivo imaging. Our data show that while zebrafish larvae can adapt to the ideal conditions for mammalian cells, human cancer cells are highly sensitive to both temperature change and transplant site. Human cells housed in slightly cooler than physiologic temperatures had a significantly altered metabolism that resulted in changes in growth, survival, and response to chemotherapy. Cancer cells xenografted into the yolk of the larvae also had reduced proliferation and drug response compared to those xenografted into the soma, in part due to the differences in metabolites available at these sites. In total, temperature and transplant site can have profound effects on xenografted cells. Standardization of zebrafish xenograft methods will enhance data reproducibility between individual laboratories.

Tributyltin (TBT) is one of the most toxic anthropogenic compounds introduced into the marine environment. Despite its global ban in 2008, TBT is still a problem of great concern due to its high affinity for particulate matter, providing a direct and potentially persistent route of entry into benthic sediments. Bioremediation strategies may constitute an alternative approach to conventional physicochemical methods, benefiting from the microorganism’s potential to metabolize anthropogenic compounds. In this work, a simple, precise and accurate static headspace gas chromatography method was developed to investigate the ability of TBT degrading microbes in sedimentary microcosms over a period of 120 days. The proposed method was validated for linearity, repeatability, accuracy, specificity, limit of detection and limit of quantification. The method was subsequently successfully applied for the detection and quantification of TBT and degrading compounds in sediment samples on day 0, 30, 60, 90 and 120 of the experiment employing the principles of green chemistry. On day 120 the concentration of TBT remaining in the microcosms ranged between 91.91 ng/g wet wt for the least effective microbial inoculant to 52.73 ng/g wet wt for the most effective microbial inoculant from a stating concentration of 100 ng/g wet wt.

Steroid analysis is important in the clinical assessment of endocrine function in health and disease. Although tandem mass spectrometry methods coupled with chromatographic separation are considered the gold standard analytical technique in this setting, enabling profiling of multiple steroids in a single sample, sample processing can be labour-intensive. Here we present a simple, efficient automated 96-well Supported Liquid Extraction method with dichloromethane/isopropanol as organic solvent, carried out on a Extrahera automated sample handler (Biotage), which completes sample preparation of 80 plasma samples (200µL) in 90 minutes. Compounds were separated on a Kinetex C18 column (150x3mm;2.6um) using a mobile phase of methanol and water (0.1% formic acid). The run time was 16 minutes on a Nexera uHPLC system (Shimadzu) with a QTrap 6500+ linear ion trap mass spectrometer (AB Sciex). Precisions ranged 8.1 to 18.1% RSD, bias -10.1-5.8%, and extraction recoveries 73.5-111.9%. LOQs ranged between 0.025–0.500 ng/mL.

A multi-tool analytical practice was used for characterization of 16^th century carpet manufactured in Cairo. Mild extraction method with hydrofluoric acid enabled isolation of intact flavonoids and their glycosides, anthraquinones, tannins and indigoids from fibre samples. High-performance liquid chromatography coupled to spectroscopic and mass spectrometric detectors was used for identification of natural dyes present in the historical samples. Weld, young fustic and brazilwood were identified as the dye sources in yellow thread samples. Red fibres have been colored with lac dye, whereas green fibre shades were obtained with indigo and weld. Tannin-containing plant material in combination with indigo and weld were used to obtain brown hue of thread. Four uncommon and thus-far unknown dye components were also found in the historical samples. These compounds probably represent unique fingerprint of dyed threads from this region. Scanning electron microscopy with energy-dispersive X-ray detector (SEM-EDS) and Fourier transformation infrared spectroscopy (FT-IR) were used for identification and characterization of substrates and mordants present in the historical carpet. Carbon and oxygen were detected in large quantities as a part of the wool protein. The presence of aluminum, iron and calcium indicated their usage as mordants. FT-IR analysis showed bands characteristic to woolen fibres and SEM micrographs definite structure of wool.

Abstract: Mass spectrometry research laboratories reported multiple probes for ambient ionization in the last years. Combining them with a mechanical moving stage enables automated sampling and imaging applications. We developed a robotic platform, which is based on RepRap 3D-printer components, and therefore easy to reproduce and to adopt for custom prototypes. The minimal step width of the Open LabBot is 12.5 μm, and the sampling dimensions (x, y, z) are 18 × 15 × 20 cm. Adjustable rails in an aluminium frame construction facilitate the mounting of additional parts such as sensors, probes, or optical components. The Open LabBot uses industry-standard G-code for its control. The simple syntax facilitates the programming of the movement. We developed two programs: 1) LABI-Imaging, for direct control via a USB connection and the synchronization with MS data acquisition. 2) RmsiGUI, which integrates all steps of mass spectrometry imaging: The creation of G-code for robot control, the assembly of imzML files from raw data and the analysis of imzML files. We proved the functionality of the system by the automated sampling and classification of essential oils with a PlasmaChip probe. Further, we performed an ambient ionization mass spectrometry imaging (AIMSI) experiment of a lime slice with laser desorption low-temperature plasma (LD-LTP) ionization, demonstrating the integration of the complete workflow in RmsiGUI. The design of the Open LabBot and the software are released under open licenses to promote their use and adoption in the instrument developers’ community.

Concentrations of six polycyclic aromatic hydrocarbons (benzanthrone, benz[a]anthracene, chrysene, fluoranthene, pyrene, and triphenylene) of soil samples from Diboll, an East Texas city (USA), were analyzed with gas chromatography-mass spectrometry (GC-MS). Samples were collected from five sites; Old Orchard Park, two heavy traffic intersections (Judd Street and Lumberjack Drive), an industrial site (West Borden Drive), and a truck stop. Acetone and dichloromethane extracts in all samples showed the presence of fluoranthene and pyrene. The sum of fluoranthene and pyrene concentrations in sites followed the order West Borden Drive > Judd Street > Lumberjack Drive > Old Orchard Park > truck stop. Concentrations of fluoranthene and pyrene were in the range 12.3 – 396.5 μg kg^-1 (ppb) and 13.6 – 209.8 μg kg^-1 ₍in dry soil₎, respectively. Benzanthrone, benz[a]anthracene, chrysene, and triphenylene concentrations were < 2 ppb levels. The higher concentrations in soils were associated with sites close to heavy traffic and vehicular emissions.

Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.

Lichen secondary metabolites are generally considered as repellent compounds for lichen feeders. Nevertheless, if the only food available consists in lichens rich in secondary metabolites, lichenophages such as Notodiscus hookeri, a gastropod native from the Possession Island, seem able to implement strategies to overcome the toxicity of these metabolites. Thus, the balance between phagostimulant nutrients and deterrent metabolites appears to play a key role in their feeding preferences. To further understand lichen-gastropod interactions, we studied the feeding behavior of Notodiscus hookeri fed exclusively with Usnea taylori, a lichen rich in usnic acid and arabitol. Snail feeding choice experiments with intact lichens vs acetone-rinsed lichens were carried out to study the influence of secondary metabolites. Simultaneously, usnic acid and arabitol were quantified and localized within the lichen thallus using HPLC-DAD-MS and in situ imaging by mass spectrometry to assess whether their spatial distribution induce preferential snail grazing. Then, no-choice feeding experiments were devised using usnic acid and arabitol embedded in artificial diet, separately or together. This case study demonstrated that the nutritional activity of N. hookerii was governed by the chemical quality of the food and primarily by nutrient availability (arabitol), despite the presence of deterrent metabolites (usnic acid).

Human milk composition is dynamic and substitute formulae are intended to mimic its protein content. The purpose of this study was to investigate the potentiality of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by multivariate data analyses as a tool to analyze peptide profiling of mammalian, human and formula milks. Breast milk samples from women at different lactation stages (2 (n = 5), 30 (n = 6), 60 (n = 5), and 90 (n = 4) days postpartum), and milk from donkeys (n = 4), cows (n = 4), buffaloes (n = 7), goats (n = 4), ewes (n = 5), and camels (n = 2) were collected. Different brands (n = 4) of infant formulae were also analyzed. Protein content (<30 kDa) was analyzed by MS and data were exported for statistical elaborations. Mass spectra for each milk closely clustered together, whereas different milk samples resulted well separated. Human samples formed a cluster in which colostrum constituted a well-defined subcluster. None of the milk formulae correlated with animal or human milk, although specifically characterized and well correlated each other. These findings propose MALDI-TOF MS milk profiling as an analytical tool to discriminate, in a blinded way, different milk types. As each formula has a distinct specificity, shifting a baby from one to another formula implies a specific proteomic exposure. These profiles may assist in milk proteomics for easiness of use and low cost consuming, suggesting that the MALDI-TOF MS pipelines may result useful for milk adulteration assessment but also for the characterization of banked milk specimens in paediatric clinical settings.

The reliable online analysis of volatile compounds in exhaled breath remains a challenge as a plethora of molecules occur in different concentration ranges (i.e. ppt to %), and need to be detected against an extremely complex background matrix. While this complexity is commonly addressed by hyphenating a specific analytical technique with appropriate preconcentration and/or preseparation strategies prior to detection, we herein propose the combination of three analytical tools based on truly orthogonal measurement principles as an alternative solution: field-asymmetric ion mobility spectrometry (FAIMS), Fourier-transform infrared (FTIR) spectroscopy-based sensors utilizing substrate-integrated hollow waveguides (iHWG), and luminescence sensing (LS). These three tools have been integrated into a single compact analytical platform suitable for online exhaled breath analysis. The analytical performance of this prototype system was tested via artificial breath samples containing nitrogen (N2), oxygen (O2), carbon dioxide (CO2) and acetone as a model volatile organic compound (VOC) commonly present and detected in breath. Functionality of the combined system was demonstrated by detecting these analytes in their respectively breath-relevant concentration range and mutually independent of each other generating orthogonal yet correlated analytical signals. Finally, adaptation of the system towards the analysis of real breath samples during future studies is discussed.

Lipid metabolism is clearly associated to Parkinson´s disease (PD). Although lipid homeostasis has been widely studied in multiple animal and cellular models as well as in blood derived from PD individuals, the cerebrospinal fluid (CSF) lipidomic profile in PD remains largely unexplored. In this study, we have characterized the CSF lipidomic imbalance between neurologically intact controls (n=10) and PD subjects (n=20). The combination of dual extraction with ultra-performance liquid chromatography-electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC-ESI-qToF-MS/MS) allowed to monitor 257 lipid species across all samples. Complementary multivariate and univariate data analysis pointed out that glycerolipids (mono-, di-, and triacylglycerides), saturated and mono/polyunsaturated fatty acids, primary fatty amides, glycerophospholipids (phosphatidylcholines, phosphatidylethanolamines), sphingolipids (ceramides, sphingomyelins), N-acylethanolamines and sterol lipids (cholesteryl esters, steroids) were significantly increased in the CSF of PD compared to control group. These results, despite the limitation of being obtained in a small population, demonstrate and extensive CSF lipid remodelling in PD, shedding new light on the deployment of CSF lipidomics as a promising tool to identify potential lipid markers as well as discriminatory lipid species between PD and other atypical parkinsonisms.

A large body of evidence has linked unhealthy eating patterns with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake in nutritional epidemiology rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching urine and plasma specimens were collected at baseline and following 2 weeks of provision of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 plasma metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV < 30%) in the majority of participants (> 75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following 2 weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high resolution MS/MS and/or co-elution after spiking with authentic standards. Overall, 3-methylhistidine and proline betaine concentrations increased consistently after participants were assigned a Prudent diet (q< 0.05) in both plasma and urine samples with a corresponding decrease in the Western diet group. Similarly, creatinine-normalized urinary imidazole propionate, hydroxypipecolic acid, dihydroxybenzoic acid, and enterolactone glucuronide, as well as plasma ketoleucine and ketovaline increased with a Prudent diet (p< 0.05) after adjustments for age, sex and BMI. In contrast, plasma myristic acid, linoelaidic acid, linoleic acid, a-linoleic acid, pentadecanoic acid, alanine, proline, carnitine and deoxycarnitine, as well as urinary acesulfame K increased among participants following a Western diet. Most metabolites were also correlated (r > ±0.30, p< 0.05) to changes in average intake of specific nutrients from self-reported diet records reflecting good adherence to assigned food provisions. Our study revealed robust biomarkers sensitive to short-term changes in habitual diet for accurate monitoring of healthy eating patterns in free-living populations, which is required for validating evidence-based public health policies for chronic disease prevention.

Ataxin-2 (ATXN2) acts during stress-responses, modulating mRNA translation and nutrient metabolism. Atxn2 knockout mice exhibit progressive obesity, dyslipidemia and insulin resistance. Conversely, the progressive ATXN2 gain-of-function due to polyGlutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2), with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-KnockIn (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin and gangliosides GM1a/GD1b, despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides, with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, of Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very-long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage, not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals, so our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode and mouse orthologs as mTORC1 inhibitors and autophagy promoters.

Metabolomics is now considered to be a wide-ranging, sensitive and practical approach to acquire useful information on the composition of a metabolite pool present in any organism, including plants. Investigating metabolomic regulation in plants is essential to understand their adaptation, acclimation and defense response to environmental stresses through the production of numerous metabolites. Moreover, metabolomics can be easily applied for the phenotyping of plants; and thus, it has great potential to be used in molecular breeding and genome editing programs to develop superior next generation crops. This review describes the recent analytical tools and techniques available to study plants metabolome, along with their significance of sample preparation using targeted and non-targeted method. Advanced analytical tools, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectroscopy (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) and matrix-assisted laser desorption/ionization (MALDI) have speed up metabolic profiling in plants. Further, we deliver a complete overview of bioinformatics tools and plant metabolome database that can be utilized to advance our knowledge to plant biology.

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative contents of free fatty acids (FFAs). Thereby, nitrogen-fixing bacteria (order Rhizobiales), often found in symbiosis with legumes, attract a special interest. Indeed, FAs impact on the structure of rhizobial nodulation factors, required for successful infection of plant root. Although the FA patterns can be addressed by GC- and LC-MS, these methods are time-consuming and suffer from compromised sensitivity, low stability of derivatives and artifacts. In contrast, MALDI-TOF-MS represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by the combination of Langmuir technology and MALDI-TOF-MS, which is featured with high sensitivity, accuracy and precision of quantification. Here we propose a step by step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. To demonstrate the analytical potential of the protocol we illustrate it by a case study – comparison the FFA metabolomes of two rhizobia species - Rhizobium leguminosarum and Sinorhizobium meliloti.

Plasma and serum are the most widely used blood-derived biofluids for metabolomics and lipidomics assays, but the isolation of these products from blood may introduce additional bias as indicated by the fact that many analytes that are present at high concentrations in blood cells cannot be measured and evaluated in those samples. Of particular concern, variable hemolysis during the pre-processing of blood products could compromise accurate and reproducible quantification. Compared with plasma or serum, whole blood may be a better alternative due to simplicity of processing. In this study, we provide a comprehensive method for quantification of the whole blood sphingolipidome and the concentrations were compared with those from plasma. Combining a single-phase extraction method with liquid-chromatography high resolution mass spectrometry (R=120, 000), assisted by alkaline hydrolysis, we were able to identify and simultaneously quantify more than 150 sphingolipids. Furthermore, most of sphingolipids remained stable after a freeze/thaw cycle. Whole blood contained a higher concentration of most sphingolipids than corresponding plasma. Moreover, individual variations in the levels of sphingolipids were lower for whole blood than plasma. These findings demonstrate that whole blood could be a better alternative to plasma, and potentially guide the evaluation of sphinglipidome for biomarker discovery.

Microsamples (collections usually less than 50 µL) have been introduced in pre-clinical, clinical, and research settings to overcome obstacles in sampling via traditional venipuncture. However, venipuncture remains the sampling gold standard for metabolic phenotyping of blood. This pre-sents several challenges in metabolic phenotyping workflows: accessibility for individuals in ru-ral and remote underserved areas (due to the need for trained personnel), the unamenable nature to frequent sampling protocols in longitudinal research (for its invasive nature), and sample col-lection difficulty in the young and elderly. Furthermore, venous sample stability may be compro-mised when temperate conditions necessary for cold-chain transport are beyond control. Alter-natively, research utilising microsamples extends phenotyping possibilities to inborn errors of metabolism, therapeutic drug monitoring, nutrition, as well as sport and anti-doping. Although the application of microsamples in metabolic phenotyping exists, it is still in its infancy, with whole blood being overwhelmingly the primary biofluid collected through the collection method of dried blood spots. Research into metabolic phenotyping of microsamples is limited; however, with advances in commercially available microsampling devices, common barriers such as volumetric inaccuracies and the ‘haematocrit effect’ in dried blood spot microsampling can be overcome. In this review, we provide an overview of the common uses and workflows for mi-crosampling in metabolic phenotyping research. We discuss the advancements in technologies, highlighting key considerations and remaining knowledge gaps for employment of microsamples in metabolic phenotyping research. Supporting the translation of research from the ‘bench to the community’.

This study evaluated the antimicrobial activity of Leptospermum scoparium (Mānuka) and Cryptomeria japonica (Sugi) essential oils and assessed the effect of seasonal chemical variation on the oils’ antimicrobial efficacies. Plate based assays were conducted to elucidate the oils’ spectrum of in vitro antimicrobial activity and to determine the oils’ minimum inhibitory concentrations (MIC) as a measure of antimicrobial efficacy. Gas chromatography – mass spectrometry was adopted to chemically profile oils distilled in different seasons. The resultant compositional information in conjunction with MIC data was used to evaluate the effect of seasonal variation on the oils’ antimicrobial efficacy. Both Mānuka and Sugi essential oils were active against all classes of target microorganisms. However, limited activity was observed against Gram-negative bacteria. The oils displayed consistent chemotypic characteristics regardless of the time of distillation. Nonetheless, there were quantitative differences in compound abundance in both essential oils. Significant differences in the MIC of Sugi essential oil was observed against target microorganisms as a result of seasonal variation in constituent abundances while Mānuka essential oil’s antimicrobial efficacy was unaffected. This study demonstrates that seasonal chemical variation is an important quality assurance parameter to consider for future application of essential oils as antimicrobial agents in consumer products.

Direct mass spectrometry-based metabolomics has been widely employed in the last years to characterize metabolic alterations underlying to Alzheimer’s disease development and progression. This high-throughput approach presents a great potential for fast and simultaneous fingerprinting of a vast number of metabolites, which can be applied to multiple biological samples such as serum/plasma, urine, cerebrospinal fluid and tissues. In this review article we present the main advantages and drawbacks of metabolomics based on direct mass spectrometry compared with conventional analytical techniques, and provide a comprehensive revision of the literature on the application of these tools in Alzheimer’s disease research.

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

The application of electrospray ionization mass spectrometry (ESI-MS) as a direct method for detecting reactive intermediates is a technique of developing importance in the routine monitoring of solution-phase reaction pathways. Here, we utilize a novel on-line photolysis ESI-MS approach to detect the photoproducts of riboflavin in aqueous solution under mildly alkaline conditions. Riboflavin is a constituent of many food products, so its breakdown processes are of wide interest. Our on-line photolysis setup allows for solution-phase photolysis to occur within a syringe using UVA LEDs, immediately prior to being introduced into the mass spectrometer via ESI. Gas-phase photofragmentation studies via laser-interfaced mass spectrometry of deprotonated riboflavin, [RFH], the dominant solution-phase species under the conditions of our study, are presented alongside the solution-phase photolysis. The results obtained illustrate the extent to which gas-phase photolysis methods can inform our understanding of the corresponding solution-phase photochemistry. We determine that the solution-phase photofragmentation observed for [RFH] closely mirrors the gas-phase photochemistry, with the m/z 241 ion being the only major condensed-phase photoproduct. Further gas-phase photoproducts are observed at m/z 255, 212, and 145. The value of exploring both the gas- and solution-phase photochemistry to characterize photochemical reactions is discussed.

The four solvent extractives obtained from aerial parts of Leucophyllum frutescens were evaluated for their Total Antioxidant Activity (TAA) by ammonium molybdate method, scavenging potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox-Equivalent Antioxidant Capacity (TEAC) assays, metal-reducing potential by Cupric Reducing Antioxidant Capacity (CUPRAC) and Ferric Reducing Antioxidant Power (FRAP) assays, Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and their biological activities. The study concluded that BULE exhibited total antioxidant activity (226.235±1.222 mg AA.Eq.gm-1 DE±S.D) by molybdate method, CHLE exhibited more scavenging potential (DPPH 209.589±8.500 mg trolox Eq.gm-1 DE±S.D and TEAC 210.166±7.954 mg trolox Eq.gm-1 DE±S.D) and reducing potential (CUPRAC 646.889±16.889 mg trolox Eq.gm-1 DE±S.D & FRAP 472.981±15.625 mg trolox Eq.gm-1 DE±S.D). Phytochemical quantification concluded high TPC by BULE (189.369±1.393 mg GA.Eq.gm-1 DE±S.D) and high TFC by CHLE (232.458±1.589 mg Qu.Eq.gm-1 DE±S.D). Strong inhibition of α-glucosidase and urease enzymes was observed by HELE (IC50 0.3321±0.007 mg.ml-1±SD) and BULE (IC50 4.09±0.357 mg.ml-1±SD) extractives, respectively. The hemolytic effect shown by hexane extract (HELE) was higher with HA50 25.545±0.927 ug.ml-1±SD whereas methanol (MELE), chloroform (CHLE), and butanol (BULE) exhibited hemolytic effects at higher concentration with HA50 400.067±1.364, 321.394±1.332, and 332.957±0.465 µg.ml-1±SD, respectively. GC-MS profiling of HELE of L. frutescens was performed for qualitative analysis. The principal phytochemicals tentatively identified by GC-MS analysis of HELE accounts for fatty acids (60.221%), lignans (17.687%), ketones (3.358%), phenols (2.584%), sesquiterpenes (1.265%), and aldehydes (0.345%).

Cyanobacteria are microorganisms able to adapt to a wide variety of environmental conditions and abiotic stresses. They produce a very large number of metabolites that can participate in the adaptation of cyanobacteria to a large range of resources such as light, temperature, or nutrient. The metabolites variation is one way to understand the physiological status and adaptation of cells. In this study, we aim to understand how the diversity and the dynamics of the whole metabolome is dependent of the growth phases and under control of abiotic factors (e.g. light intensity and temperature). The cyanobacteria Aliinostoc sp. PMC 882.14 was selected for its large number of biosynthetic gene clusters. Metabolomes were analyzed by using mass spectrometry (qTOF-MS/MS) combined with untargeted analysis to investigate the metabolite dynamics. Significant variations were characterized between exponential and stationary phases, whatever the culture conditions (“control”, “higher light”, or “higher temperature”). ”Higher light” and “higher temperature” favored the synthesis of metabolites belonging to the same molecular families. Among highly regulated metabolites, we observe the presence of mycosporine-like amino acids (MAAs), and various variants of somamides, microginins, and microviridins. Through Aliinostoc sp. PMC 882.14, this study shows the importance of knowing the physiological state of cyanobacteria for comparative global metabolomics and questions the regulation processes involve into metabolite families production. Our results also open up new perspectives in the context of the production of targeted bioactive metabolites.

Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.

Primates are traditionally considered to have a poor sense of smell. However, olfaction is important for non-human primates as demonstrated by conspicuous scent-marking behaviours in lemurs. We studied two family groups (N=4) of crowned lemurs (Eulemur coronatus) housed at Colchester and Twycross zoos (UK) by combining behavioural observations and chemical analyses of scent-marks. We recorded observations of olfactory behaviours for 201 h using instantaneous scan sampling. We investigated the volatile compounds of ano-genital odour secretions using solid-phase microextraction and gas chromatography-mass spectrometry. Males scent-marked most frequently, displaying ano-genital marking for allomarking, head marking for countermarking and wrist marking in specific areas of the enclosure. Females displayed ano-genital marking predominantly on feeding devices. We detected a total of 38 volatile components in all male ano-genital scent-marks and 26 in all female samples of ano-genital odour secretions, including a series of esters, aldehydes, ketones, alcohols, terpenes, volatile fatty acids and hydrocarbons that have been identified in odour profiles of other primates. In conclusion, we found sexual dimorphism in crowned lemurs. Male head and wrist marking behaviours might play defensive territorial functions, while ano-genital marking would be related to socio-sexual communication as chemical mate-guarding. Female ano-genital marking might be involved in resource defense.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggests that cancer-derived EVs may carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in breast cancer EVs compared to vesicles secreted from benign breast epithelial cells. Total EV integrin levels, including the quantity of integrins α2, αv, β4, and β5 correlate with breast tumor stage. In particular, integrin α2 also largely reflects progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in cancer diagnosis and prediction of tumor stage. Differential expression of integrins across cancer cells, and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.

Data independent acquisition - mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on Arabidopsis sample with spike-in of known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 59.4% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the 6-protein mix spike-in. In cDIA only 5 out of the 6-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus), identified 268 proteins with age-related structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I, II and V α-chains (COL1A2, COL2A1, COL5A1), were located within their triple helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance in ageing. Several proteins (COL5A1, COL2A1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young, suggesting that some ageing mechanisms may act locally within tissues. This study not only provides evidence of age-related changes in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF to identify potential protein biomarkers of localised tissue remodelling.

Both ricin and R. communis agglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. LC-HRMS was used to verified the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, 6 and 5 peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

1) Background: Recently, illegal abuse of γ-hydroxybutyric acid (GHB) has increased in drug-facilitated crimes, but determination of GHB exposure and intoxication is difficult due to rapid metabolism of GHB. Its biochemical mechanism has not been completely investigated. And metabolomic study by polyamine profile and pattern analyses was not performed in rat urinefollowing intraperitoneal injection with GHB. 2) Methods: Polyamine profiling analysis by gas chromatography-mass spectrometry combined with star pattern recognition analysis was performed in this study. Multivariate statistical analysis was used to evaluate discrimination between control and GHB administration groups. 3) Results: Six polyamines were determined in control, single and multiple GHB administration groups. Star pattern showed distorted hexagonal shapes with characteristic and readily distinguishable patterns for each group. N¹-Acetylspermine (p < 0.001), putrescine (p <0.006), N¹-acetylspermidine (p <0.009), and spermine (p < 0.027) were significantly increased in single administration group but were significantly lower in the multiple administration group than in the control group. N¹-Acetylspermine was the main polyamine for discrimination between control, single and multiple administration groups. Spermine showed similar levels in single and multiple administration groups. 4) Conclusions: The polyamine metabolic pattern was monitored in GHB administration groups. N¹-Acetylspermine and spermine were evaluated as potential biomarkers of GHB exposure and addiction.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. After the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“Oral Turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights on the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations of the D-ring.

Abstract Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains which associates with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in cellular trafficking of different proteins, EV cargo sorting and vesicles formation. We have preciously shown that CD63 is important in LMP1 trafficking to EVs and this also affects LMP1 mediated intracellular signaling including MAPK/ERK, NF-κB and mTOR activation. Using the BioID combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as proximal interacting newtwork of proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism and transportation. The CD63 only interactome was enriched in Rab GTPases, SNARE proteins and sorting nexins while adding LMP1 into the interactome increased presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of proteins enrichment from protein localization and vesicle mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTor, Nedd4L and PP2A indicating formation of a multiprotein complex with CD63 thereby potentially regulating LMP1 dependent mTor signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into network of partners required for endocytic trafficking, extracellular vesicle cargo sorting, formation and secretion.

Objective: Lipids are secreted into milk as bilayer-coated structures: milk lipid globules (MLGs). Adipophilin (ADRP) and perilipin 3 (TIP47) are associated with MLGs in human breast milk; however, the role of these proteins in milk lipid secretion is not fully understood. The aim of the study was to investigate levels of ADRP, TIP47 and total lipid content in human breast milk, their mutual correlations and dynamics during lactation. Research Methods & Procedures: Milk samples from 22 healthy lactating women (Caucasian, Central European) were collected at five time points during lactation (1–3, 12–14, 29–30, 88–90 and 178–180 days postpartum). Mass spectrometry-based method was used for quantification of ADRP and TIP47 in the samples. The gravimetric method was used to determine milk total lipid content. Results: We observed distinctive trends in ADRP, TIP47 levels and lipid content in human breast milk during the first 6 months of lactation. We also found a significant association between lipid content and ADRP, lipid content and TIP47, and ADRP and TIP47 concentrations in breast milk at all sampling points. Moreover, we derived an equation for estimating the mean lipid content of milk based on ADRP concentration in human breast milk. Conclusions: A mass spectrometry-based method was developed for quantifying ADRP and TIP47 in human breast milk. Strong mutual correlations were found between ADRP, TIP47 and total lipid content in human breast milk.

In this study, the use of recycled diatomaceous earth as the extraction phase in a the solid phase microextraction (SPME) technique for the determination of polycyclic aromatic hydrocarbons (PAHs) in river water samples, with separation/detection performed by gas chromatography-mass spectrometry (GC-MS), is proposed. The optimized extraction conditions are extraction time 70 min at 80 ºC with no addition of salt. The limits of quantification were close to 0.5 μg L-1 with RSD values lower than 25% (n = 3). The linear working range was 0.5 μg L-1 to 25 μg L-1 for all analytes. The method was applied to samples collected from the Itajaí River (Santa Catarina, Brazil) and the RSD values for repeatability and reproducibility were lower than 15% and 17%, respectively. The efficiency of the recycled diatomaceous earth fiber was compared with that of commercial fibers and good results were obtained, confirming that this is a promising option for use as the extraction phase in SPME.

Two-dimensional mass spectrometry (2D MS) is a method for tandem mass spectrometry that relies on manipulating ion motions to correlate precursor and fragment ion signals. 2D mass spectra are obtained by performing a Fourier transform in both the precursor ion mass-to-charge ratio (m/z) dimension and the fragment ion m/z dimension. The phase of the ion signals evolves linearly in the precursor m/z dimension and quadratically in the fragment m/z dimension. This study demonstrates that phase-corrected absorption mode 2D mass spectrometry improves signal-to-noise ratios by a factor of 2 and resolving power by a factor of 2 in each dimension compared to magnitude mode. Furthermore, phase correction leads to an easier differentiation between ion signals and artefacts, and therefore easier data interpretation.

One of the richest natural sources of resveratrol - the rhizome of Reynoutria japonica in East Asia is a well-known traditional herb (Hu zhang) used in various inflammatory diseases, infections, skin diseases, scald, hyperlipidemia. Although, it has been recently included in the European Pharmacopoeia, still in Europe is an untapped resource. Some of the therapeutic effects are likely to be influenced by its antioxidant properties and this in turn is frequently associated with a high stilbene content. However, some literatures suggested that other compounds than stilbenes may add to the total antioxidant capacity. Hence, the aim of this research was to examine rhizomes of R. japonica and less studied, morphologically similar species, R. sachalinensis and R. x bohemica for their phytochemical composition and antioxidant activity and to clarify the relationship between the antioxidant activity and compounds by statistical methods. HPLC/UV/ESI-MS studies of three Reynoutria species revealed 171 compounds comprising stilbenes, carbohydrates, procyanidins, flavan-3-ols, anthraquinones, phenylpropanoids, lignin oligomers, hydroxycinnamic acids, naphthalenes and their derivatives. Our studies confirmed the presence of procyanidins with high degree of polymerization, up to decamers in the rhizomes of R. japonica and brings new data on the presence of these compounds in other Reynoutria species. A procyanidin trimer digallate was described for the first time in the studied plants. Moreover, we suggested a presence of new for these species, dianthrone glycosides and previously unrecorded phenylpropanoid disaccharide esters and hydroxycinnamic acid derivatives, mainly in R. sachalinensis. Furthemore, compounds tentatively annotated as lignin oligomers were observed for the first time in studied species. The rhizomes of all Reynoutria species exhibited strong antioxidant activity. Statistical analysis demonstrated that proanthocyanidins should be considered as important contributors to the total antioxidant capacity.

Almond is worldwide consumed and renowned as a valuable healthy food. In spite of this, it is also a potent source of allergenic proteins able to trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials, were evaluated by total protein quantification, ELISA assay and protein profiling by electrophoresis-based separation (SDS-PAGE). The autoclaving alone was found to weakly affect almond proteins stability, despite what observed for the combination of hydration and autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated sample, and in a final negligible immunoreactivity, as well. The final SDS-PAGE protein pattern recorded for almonds hydrated and autoclaved disclosed significant changes. In addition, the same samples were further submitted to in vitro simulated gastro-duodenal (GI) digestion to evaluate potential changes induced by these processing on allergens digestibility. Digestion products were identified by HPLC-HRMS/MS analysis followed by software-based data mining, and complementary information were provided by analyzing the proteolytic fragments lower that 6 kDa in size. The autoclave based treatment was found not to alter the allergens digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to the combination of prehydration and autoclaving. Finally, the residual immunoreactivity of the GI resistant peptides was investigated in-silico by bioinformatic tools, confirming that by following both approaches, no epitopes survived the almond digestion, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

One of the research priorities in atmospheric chemistry is to advance our understanding of heterogeneous reactions and their effect on the composition of the troposphere. Chemistry on aqueous surfaces is particularly important in this regard because of their ubiquity and expanse. They range from the surfaces of oceans (360 million km²), cloud and aerosol drops (~10 trillion km²) to the fluids lining the human lung (~200 m²). Typically, ambient air contains reactive gases that may affect human health, influence climate and participate in biogeochemical cycles. Despite their importance, reactions between gases and solutes at air-aqueous interfaces are not well understood. New, surface-specific techniques are required that detect and identify the intermediates and products of such reactions as they happen on liquids. This is a tall order because genuine interfacial reactions are faster than mass diffusion into bulk liquids, and may produce novel species at low concentrations. Herein, we review evidence that validates online pneumatic ionization mass spectrometry of liquid microjets dosed by reactive gases as a technique meeting such requirements. Next, we call attention to results obtained by this approach on reactions of ozone, nitrogen dioxide and hydroxyl radicals with various solutes on aqueous surfaces. The overarching conclusion is that the outermost layers of aqueous solutions are unique media, where equilibria shift and reactions proceed faster than, in some cases along different pathways from the bulk liquids. The fact that the rates and mechanisms of reactions at air-aqueous interfaces may not be deduced from experiments in bulk liquids opens new conceptual frameworks and lines of research, and adds an overlooked dimension to atmospheric chemistry.

The present study evaluates the bioremediation potential of indigenous bacterial species isolated from dye-contaminated soil samples from small dyeing outlet located in Ilorin. The water pollution index was estimated based on the physicochemical characteristics and heavy metal concentrations of the raw (Day 0) and treated textile wastewater such as pH, biochemical oxygen demand-5, chemical oxygen demand, total suspended solids and total dissolved solid with mean values of 8.85±0.45 mg/L, 1200±21.3 mg/L, 2440±31.3 mg/L, 1660±17.2 mg/L and 2650±28.1 mg/L respectively, similarly, Lead was the most abundant heavy metal detected in the sample while Cadmium concentration was the lowest with the mean values of 3.52±0.00 mg/L and 2.18±0.00 mg/L respectively. The bacterial strain with highest dye decolorization capacity was screened and identified as Bacillus licheniformis ZUL012.The isolate was consequently used for the bioremediation of the wastewater over a period of 10 days. The results showed an incredible reduction in the physiochemical characteristics and heavy metal concentrations of the textile wastewater in the following ranges (8.85-6.55), (1200-300) mg/L, (2440-518) mg/L, (1660-666) mg/L and (2650-920) mg/L with the highest removal efficiency of 75 %, 78 %, 60%, 65%, recorded for biochemical oxygen demand, chemical oxygen demand, total suspended solid, total dissolved solid, respectively while that of heavy metals such as lead, cadmium, chromium and nickel were 80 %, 60 %, 67 %, 72 % reduction, respectively. Laccase and Azoreductase activities tend to decrease as the pH gradually moved towards acidic condition during the bioremediation process. Toxicity of the treated effluent was assessed using Maize and Bean seed germination test. Conclusively, these research findings can serve as a framework for the outlet design of wastewater treatment plant for local textile outlets.

Human pluripotent stem cells (hPSCs) have widespread potential biomedical applications. There is a need for large-scale in vitro production of hPSCs, and optimal culture methods are vital in achieving this. Physiological oxygen (2% O2) improves key hPSCs attributes, including genomic integrity, viability, and clonogenicity, however, its impact on hPSC metabolism remains un-clear. Here, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) was used to detect and quantify metabolic Volatile Organic Compounds (VOCs) in the headspace of hPSCs and their differentiated progeny. hPSCs were cultured in either 2% O2 or 21% O2. Media was collected from cell cultures and transferred into glass bottles for SIFT-MS measurement. The VOCs acetaldehyde and dimethyl sulfide (DMS)/ethanethiol were significantly increased in undifferentiated hPSCs compared to their differentiating counterparts, and these observations were more apparent in 2% O2. Pluripotent marker expression was consistent across both O2 concentrations tested. Transcript levels of ADH4, ADH5, and CYP2E1, encoding enzymes involved in converting ethanol to acetaldehyde, were upregulated in 2% O2, and chemical inhibition of ADH and CYP2E1 decreased acetaldehyde levels in hPSCs. Acetaldehyde and DMS/ethanethiol may be indicators of altered metabolism pathways in physiological oxygen culture conditions. The identification of non-destructive biomarkers for hPSC characterization has the potential to facilitate large-scale in vitro manufacture for future biomedical application.

Man-made shallow fishponds in the Czech Republic have been facing a high eutrophication since 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physico-chemical properties of water and its aquatic community composition leading to harmful algal bloom formation. In our current study, we have characterised the phytoplankton community across three hypertrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprised of non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography tandem mass spectrometry (LC–MS/MS) data coupled with dereplication strategy. This MS networking approach coupled with dereplication on online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin and epidolastatin12. We have applied the binary logistic regression to estimate the CNPs producer by correlating the GNPS data with the species abundance. Usage of The combination of molecular networking and dereplication on online global natural product social networking (GNPS) web platform has proved as a valuable approach for rapid and simultaneous detection of high number of peptides, and rapidly assessing the risk for harmful bloom.

Through its suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack". Instead, they exploit all the constituents of the haystack. This new form of analytical methods is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing needs to be propagated for the benefit of ones associated in academic research, commercial development, and official control. This paper addresses the frequently asked questions around notations and terminologies surrounding NTMs. The widespread development and adoption of these methods also necessitates the need to develop approaches to NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap to approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions thereof.

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the TCA cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxo acid dehydrogenase complexes, their function and regulation.

Leaf mechanical wounding triggers a rapid, within minutes, release of a blend of volatile organic compounds (VOCs). Wounding-induced VOC blend is mainly composed of oxygenated ubiquitous stress volatiles such as methanol and volatile products of lipoxygenase (LOX) pathway (mainly C5 and C6 alcohols and aldehydes and their derivatives), but also includes multiple minor VOCs that collectively act as infochemicals inducing defences in non-damaged plant leaves, neighbouring plants and attracting herbivore enemies. Till present, interspecific variability of the rate of induction and magnitude of wounding-induced emissions, and the extent to which plant structural traits and physiological activity alter these emissions are poorly known. Particularly scarce is the information of the induced emissions in tropical agricultural plant species despite their economic importance and large area of cultivation at regional to global scales. We chose five tropical crops with varying photosynthetic activity and leaf structural characteristics: Abelmoschus esculentus, Amaranthus cruentus, Amaranthus hybridus, Solanum aethiopicum and Telfairia occidentalis to characterize the kinetics and magnitude of wounding-induced emissions, hypothesizing that the induced emission response is greater and faster in physiologically more active species with greater photosynthetic activity than in less active species. Rapid highly repeatable leaf wounds (12-mm cuts) were generated by a within-leaf-chamber cutting knife. Wounding-induced VOC emissions were measured continuously with a proton-transfer reaction time-of-flight mass spectrometer and gas-chromatography mass spectrometry was used to separate isomers. Twenty-three ion VOCs and twelve terpenoid molecule structures were identified, whereas ubiquitous stress volatiles methanol (on average 40% of total emissions), hexenal (24%), and acetaldehyde (11%) were the main compounds across the species. Emissions of low-weight oxygenated compounds (LOC, 70% of total), and LOX products (29%) were positively correlated across species, but minor VOC components, monoterpenoids and benzenoids were negatively correlated with LOC and LOX, indicating a reverse relationship between signal specificity and strength. There was a large interspecific variability in the rate of induction and emission magnitude, but the hypothesis of a stronger emission response in physiologically more active species was only partly supported. In addition, the overall emission levels were somewhat lower with different emission blend compared to the data reported for wild species, as well as different shares for the VOCs in the blend. The study demonstrates that wounding-dependent emissions from tropical agricultural crops can significantly contribute to atmospheric volatiles, and these emissions cannot be predicted based on current evidence of wild plant model systems.

The ‘inverse problem’ of mass spectrometric molecular identification (‘given a mass spectrum, calculate/predict the 2D structure of the molecule whence it came’) is largely unsolved, and is especially acute in metabolomics where many small molecules remain unidentified. This is largely because the number of experimentally available electrospray mass spectra of small molecules is quite limited. However, the forward problem (‘calculate a small molecule’s likely fragmentation and hence at least some of its mass spectrum from its structure alone’) is much more tractable, because the strengths of different chemical bonds are roughly known. This kind of molecular identification problem may be cast as a language translation problem in which the source language is a list of high-resolution mass spectral peaks and the ‘translation’ a representation (for instance in SMILES) of the molecule. It is thus suitable for attack using the deep neural networks known as transformers. We here present MassGenie, a method that uses a transformer-based deep neural network, trained on ~6 million chemical structures with augmented SMILES encoding and their paired molecular fragments as generated in silico, explicitly including the protonated molecular ion. This architecture (containing some 400 million elements) is used to predict the structure of a molecule from the various fragments that may be expected to be observed when some of its bonds are broken. Despite being given essentially no detailed nor explicit rules about molecular fragmentation methods, isotope patterns, rearrangements, neutral losses, and the like, MassGenie learns the effective properties of the mass spectral fragment and valency space, and can generate candidate molecular structures that are very close or identical to those of the ‘true’ molecules. We also use VAE-Sim, a previously published variational autoencoder, to generate candidate molecules that are ‘similar’ to the top hit. In addition to using the ‘top hits’ directly, we can produce a rank order of these by ‘round-tripping’ candidate molecules and comparing them with the true molecules, where known. As a proof of principle, we confine ourselves to positive electrospray mass spectra from molecules with a molecular mass of 500Da or lower, including those in the last CASMI challenge (for which the results are known), getting 49/93 (53%) precisely correct. The transformer method, applied here for the first time to mass spectral interpretation, works extremely effectively both for mass spectra generated in silico and on experimentally obtained mass spectra from pure compounds. It seems to act as a Las Vegas algorithm, in that it either gives the correct answer or simply states that it cannot find one. The ability to create and to ‘learn’ millions of fragmentation patterns in silico, and therefrom generate candidate structures (that do not have to be in existing libraries) directly, thus opens up entirely the field of de novo small molecule structure prediction from experimental mass spectra.

Over the past decade, the One Strain Many Compounds (OSMAC) approach has been established for silent gene cluster activation and elicitation of secondary metabolite production, but so far the full secondary metabolome of a biosynthetically promising bacterium has not been elucidated yet. Here, we investigate the ability of seven categories of OSMAC conditions to enhance the diversity of new mass features from bacterial strains with little literature coverage but high biosynthetic potential. The strains Bacillus. amyloliquefaciens DSM7, Corallococcus. coralloides DSM2259, Pyxidicoccus. fallax HKI727, Rhodococcus. jostii DSM44719, and Streptomyces. griseochromogenes DSM40499 were selected after genome mining with antiSMASH. After cultivation under OSMAC conditions, the generated extracts were subjected to LC-MS and MZmine analysis to determine new mass features and evaluate the tested culture conditions. 4 predicted compounds, bacillibactin, desferrioxamine B, myxochelin A, and surfactin, were identified and up to 147 new mass features were detected in the generated extracts, which greatly surpasses the number of predicted gene clusters. Among the new mass features are bioactive compounds that were so far unreported for the strains such as cyclo-(Tyr-Pro) from DSM7 and nocardamin from DSM2259. Furthermore, the tested culture conditions were evaluated regarding their suitability for the generation of new mass features from the selected strains and promising new starting points for further screenings are postulated. Especially culture conditions with little prior literature coverage are responsible for the activation of predicted gene clusters

The aquaculture industry has become a sustainable provider of food to humans. Remaining challenges include disease issues, as well as ethical concerns for discomfort and stress among farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid-liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g and the limit of quantification was 0.34 ng/g, and the method was linear (R2>0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a CV of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

In this paper, experiences of the last 20 years with the PERALS-technique are described. PERALS stands for Photo Electron Rejecting Alpha Liquid Scintillation. This LSC-technique was developed by Jack McDowell in the 70ies and is a powerful technique for the analyses of many natural alpha nuclides. The principle is based on a selective extraction of the radionuclide from the water phase by means of a complexing or ion pair reagent. The extractant contains also a suitable cocktail for the scintillation counting. Therefore, the extract can be analysed directly after the extraction step. After removing quenchers, such as oxygen, and the proper setting of a pulse shape discriminator, alpha pulses can be counted with a photomultiplier. The paper describes the development of robust analysis schemes for the determination of traces of polonium, radon, radium, strontium, thorium, uranium and other actinides in water samples (groundwater, rain water, river water, drinking water, mineral water, sea water).

Alterations in the carnitine shuttle system may be an indication of the presence of cancer. As such, in-depth analyses of this pathway in different malignant tumors could be important for the detec-tion and treatment of this disease. The current study aims to assess the profiles of carnitine and acylcarnitines in gliomas with respect to their grade, the presence of isocitrate dehydrogenase (IDH) mutations, and 1p/19q co-deletion. Brain tumors obtained from 19 patients were sampled on-site using solid-phase microextraction (SPME) immediately following excision. Analytes were desorbed and then analyzed via liquid chromatography-high resolution mass spectrometry. The results showed that SPME enabled the extraction of carnitine and 22 acylcarnitines. An analysis of the correlation factor revealed presence of two separate clusters: short-chain and long chain car-nitine esters. Slightly higher carnitine and acylcarnitine concentrations were observed in the higher malignancy tumor samples (high vs low grade), and in those samples with worse projected clinical outcomes (without vs with IDH mutation; without vs with 1p/19q co-deletion). Thus, the proposed chemical biopsy approach offers a simple solution for on-site sampling that enables sample preservation, thus supporting comprehensive multi-method analyses.

Proteomic technology has improved at a staggering pace in recent years, with even practitioners challenged to keep up with new methods and hardware. The most common metric used for method performance is the number of peptides and proteins identified. While this metric may be helpful for proteomics researchers shopping for new hardware, this is often not the most biologically relevant metric. Biologists often utilize proteomics in the search for protein regulators that are of lower relative copy number in the cell. In this review, I re-evaluate untargeted proteomics data using a simple graphical representation of the absolute copy number of proteins present in a single cancer cell as a metric. By comparing single shot proteomics data to the coverage of the most in-depth proteomic analysis of that cell line acquired to-date we can obtain a rapid metric of method performance. Using a simple copy number metric allows visualization of how proteomics has developed in both sensitivity and overall dynamic range when using both relatively long and short acquisition times. To enable reanalysis beyond what is presented here, two available web applications have been developed for single and multi-experiment comparisons with reference protein copy number data for multiple cell lines and organisms.

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

Isobaric ions having the same mass-to-charge ratio cannot be separately identified by mass spectrometry (MS) alone, but this limitation can be overcome using hydrogen-deuterium exchange (HDX) in microdroplets. Because isobaric ions may contain a varied number of exchangeable sites and different types of functional groups, each one produces a unique MS spectral pattern after droplet spray HDX without the need for MS/MS experiments or introduction of ion mobility measurements. As an example of the power of this approach, isobaric ions in urinary metabolic profiles are identified and used to distinguish between healthy individuals and those having bladder cancer.

About 50% of colorectal cancer patients develop liver metastases. Patients with metastatic colorectal cancer have 5-year survival rates below 20% despite new therapeutic regimens. Tumor heterogeneity has been linked with poor treatment response and clinical outcome, but was so far mainly studied via bulk genomic analyses. In this study we performed spatial proteomics via MALDI mass spectrometry imaging on six patient-matched CRC primary tumor and liver metastases to characterize interpatient, intertumor and intratumor hetereogeneity. We found several peptide features that were enriched in vital tumor areas of primary tumors and liver metastasis and tentatively derived from tumor cell specific proteins such as annexin A4 and prelamin A/C. Liver metastases of colorectal cancer showed higher heterogeneity between patients than primary tumors while within patients both entities show similar intratumor heterogeneity sometimes organized in zonal pattern. Together our findings give new insights into the spatial proteomic heterogeneity of primary CRC and patient-matched liver metastases.

In the recent years, Cannabis and hemp-based products have become increasingly popular for various applications ranging from recreational use, edibles, beverages to health care products and medicines. The rapid detection and differentiation of phytocannabinoids is, therefore, essential to assess the potency, therapeutic and nutritional values of cannabis cultivars. Here, we implemented the SpiderMass technology for the in vivo detection of cannabidiol acid (CBDA) and tetrahydrocannabinol acid (THCA) and other endogenous organic plant compounds to access distribution gradients within the plants and differentiate cultivars. The SpiderMass system is composed of an IR- laser handheld microsampling probe connected to the mass spectrometer through a transfer tube. The analysis was performed in situ on different plant organs from freshly cultivated Cannabis plants in only a few seconds. SpiderMass analysis easily discriminated the two acid phytocannabinoid isomers by MS/MS and the built statistical models differentiated between four Cannabis cultivars. Different abundancies of acid phytocannabinoids were also found along the plant as well as between different cultivars. All together, these results introduce the direct analysis by SpiderMass as a compelling analytical alternative for forensic and hemp industrial analysis.

The naturally occurring dipeptide carnosine (β alanyl L histidine) specifically attenuates tumor growth. Here, we asked whether other small imidazole containing compounds also affect viability of tumor cells without affecting non-malignant cells, and whether formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L alanyl L histidine (LA-LH), ß alanyl L alanine, L histidine, histamine, imidazole, β alanine and L alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L histidine and formation of histamine was investigated by High Performance Liquid Chromatography coupled Mass Spectrometry. Whereas carnosine and LA LH inhibited tumor cell growth with minor effects on fibroblasts, L-histidine, histamine and imidazole affected viability in both cell types. Compounds without imidazole moiety did not diminish viability. In the presence of LA LH but not in the presence of carnosine a significant rise of intracellular amounts of histidine was detected in all cells. Formation of histamine was not detectable in the presence of carnosine, LA LH or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA LH. Despite histamine had a strong effect on cell viability, formation of histamine is not responsible for the effects on cell viability of carnosine, LA LH and histidine.

The prevalence of food allergy has increased over the last decades and consequently the food labeling policies have improved over the time in different countries to regulate allergen presence in foods. In particular, Reg 1169 in EU mandates the labelling of 14 allergens whenever intentionally added to foods, but the inadvertent contamination by allergens still remains uncovered topic. In order to warn consumers on the risk of cross-contamination occurring in certain categories of foods, a precautionary allergen labelling system has been put in place by food industries on voluntary basis. In order to reduce the overuse of PAL, reference doses and action limits have been proposed by the VITAL project representing a guide in this jeopardize scenario. Development of sensitive and reliable mass spectrometry methods are therefore of paramount importance in this regard to check the contamination levels in foods. In this paper we describe the development of a managed time MRM method based on a triple quadrupole platform for milk and egg quantification in processed food. The method was in house validated and allowed to achieve levels of proteins lower than 0.2mg of total milk and egg proteins respectively in cookies, challenging the doses recommended by VITAL. The method was finally applied to cookies labeled as milk and egg-free. This method could represent in perspective a promising tool to be implemented along the food chain to detect even tiny amounts of allergens contaminating food commodities.

Heterogeneous distribution of delivery or treatment modalities within the tumor mass is a crucial limiting factor for a vast range of theranostic applications. Understanding the interactions between a nanomaterial and the tumor microenvironment will help to overcome challenges associated with tumor heterogeneity as well as the clinical translation of nanotheranostic materials. This study aims to evaluate the influence of protein surface adsorption on gold nanoparticle (GNP) biodistribution using high resolution Computed Tomography (CT) preclinical imaging in C57BL/6 mice harboring Lewis Lung Carcinoma (LLC) tumors. LLC provides a valuable model for study due to its highly heterogenous nature which makes drug delivery to the tumor challenging. By controlling the adsorption of proteins on the GNP surface, we hypothesize that we can influence the intratumoral distribution pattern and particle retention. We performed an in vitro study to evaluate the uptake of GNPs by LLC cells and an in vivo study to assess and quantify the GNP biodistribution by injecting concentrated GNPs citrate stabilized or passivated with Bovine Serum Albumin (BSA) intratumorally into LLC solid tumors. Quantitative CT and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) results both confirm the presence of particles in the tumor 9 days post-injection (n=8 mice/group). Significant difference is highlighted between citrate-GNP and BSA-GNP groups (**p < 0.005, Tukey’s multiple comparisons test), confirming the protein corona of GNPs modifies intratumoral distribution and retention of the particles. In conclusion, our investigations show that the surface passivation of GNPs influences the mechanism of cellular uptake and intratumoral distribution in vivo highlighting the spatial heterogeneity of the solid tumor.

In the matrix of bacteria/mitochondria/chloroplasts, Lon acts as the degradation machine for soluble proteins. In stress periods, however, proteostasis and survival depend on the strongly conserved Clp/Hsp100 family. Currently, the targets of ATP-powered unfoldases/disaggregases ClpB and ClpX, and of peptidase ClpP heptameric rings, are still unclear. Trapping experiments and proteome profiling in multiple organisms triggered confusion, so we analyzed the consistency of ClpP-trap targets in bacteria. We also provide meta-analyses of protein interactions in humans, to elucidate where Clp family members are enriched. Furthermore, meta-analyses of mouse complexomics are provided. Genotype-phenotype correlations confirmed our concept. Trapping, proteome, and complexome data retrieved consistent coaccumulation of ClpXP with GFM1 and TUFM orthologs. ClpX shows broad interaction selectivity encompassing mitochondrial translation elongation, RNA granule, and nucleoid; ClpB preferentially attaches to mitochondrial RNA granule and translation initiation components; ClpP is enriched with them all, and associates with release/recycling factors. Mutations in ClpP cause Perrault syndrome, with phenotypes similar to defects in mtDNA/mtRNA. Thus, we propose that ClpB and ClpXP are crucial to counteract misfolded insoluble protein assemblies that contain nucleotide-phosphates. This insight is relevant to improve ClpP-modulating drugs that block bacterial growth, and for the treatment of human infertility, deafness and neurodegeneration.

Paenibacilli are efficient producers of potent agents against bacterial and fungal pathogens, which are of great interest both for therapeutic applications in medicine as well as in agrobiotechnol-ogy. Lipopeptides produced by such organisms play a major role in their inactivation potential. In this work we investigated two lipopeptide complexes, the fusaricidins and the polymyxins, produced by Paenibacillus polymyxa strains DSM 32871 and M1 by MALDI-TOF mass spectrometry. The fusaricidins show potent antifungal activities and are distinguished by an unusual variabil-ity. For strain DSM 32871 we identified numerous yet unknown variants mass spectrometrically. DSM 32871 produces polymyxins of type E (colistins), while M1 forms polymyxins P. For both strains novel, but not yet completely characterized polymyxin species were detected, which pos-sibly are glycosylated. These compounds may be of interest therapeutically, because polymyxins attain increasing attention as last-resort antibiotics against multiresistant pathogenic Gram-negative bacteria. In addition, the volatilomes of DSM 32781 and M1 were investigated with a GC-MS approach using different cultivation media. Production of volatile organic com-pounds (VOCs) was strain and medium dependent. In particular, strain M1 manifested as an effi-cient VOC-producer that exhibited formation of 25 volatiles in total. A characteristic feature of Paenibacilli is the formation of volatile pyrazine derivatives.

Seafood is considered one of the main food allergen sources by the European Food Safety Authority (EFSA). It comprises several distinct groups of edible aquatic animals including fish and shellfish such as crustacean and mollusks. Recently the EFSA recognized the high risk of food allergy over the world and established the necessity of developing new methodologies for its control. Consequently, accurate, sensitive and fast detection methods for seafood allergy control and detection in food products are highly recommendable. In this work, we present a comprehensive review of the applications of the proteomics methodologies for the detection and quantification of seafood allergens. For that, two consecutive proteomics strategies (Discovery and Targeted Proteomics) applied for the study and control of seafood allergy are reviewed in detail. In addition, future directions and new perspectives were also provided.

Leishmania infantum is causative of visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, lethal if untreated. Few years ago, re-sequencing and de novo assembling of the L. infantum (strain JPCM5) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we have performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2,352 proteins based on the search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We have detected many proteins belonging to organelles such as glycosomes, mitochondria or flagellum, as well as many metabolic enzymes, and a large number of putative RNA binding proteins and molecular chaperones. Moreover, we listed the proteins presenting post-translational modifications, such as phosphorylations, acetylations and methylations, among others. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding has allowed to correct annotations, leading to N-terminal extension of protein sequences, and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics and transcriptomics has resulted in a powerful combination for improving the L. infantum reference genome annotation.

Despite the increasing popularity of liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated method for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids, however, the sample complexity and purity complex lipid extract quantification in lipidomics experiments could be impact quantification accuracy. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Pure lipids are characterized by CH-and C=O-stretching vibrations on FTIR, with quantitative range of 40–3000 ng and a limit of detection of 12 ng. Sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification by CH-stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH-stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. We evaluated LiQ score as a rapid sample quality control method by comparing to total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. Finally, the uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be &lt;10%. In summary, this study identified key parameters for accurate FTIR-based quantification of complex lipid mixture, and developed a rapid workflow requiring only 1 µL of MS-ready sample and &lt; 5 minutes for routine lipidomics sample quality and quantity assessment.

Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterized cytoskeleton-associated protein CAP5.5 as a probe we identified CAP50 (Tb927.11.2610). This protein colocalizes with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganization of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterized, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.

Alkali-containing submicron particles were measured continuously during three months, including late winter and spring seasons in Gothenburg, Sweden. The overall aims were to characterize the ambient concentrations of combustion-related aerosol particles and to address the importance of local emissions and long-range transport for the atmospheric concentrations in the urban background environment. K and Na concentrations in the PM1 size range were measured by an alkali aerosol mass spectrometer (Alkali-AMS) and a cluster analysis was conducted. Local meteorological conditions and some other data sets were obtained, and back trajectory analyses and chemical transport model (CTM) simulations were included for the evaluation. The Alkali-AMS cluster analysis indicated three major clusters: 1) biomass burning origin, 2) mixture of other combustion sources, and 3) marine origin. Low temperatures and low wind speed conditions correlated with high concentrations of K-containing particles, mainly due to regional emissions from residential biomass combustion; transport of air masses from continental Europe also contribute to cluster 1. The CTM results indicate that open biomass burning in the eastern parts of Europe may have contributed substantially to high PM2.5 concentrations (and to cluster 1) during an episode in late March. According to the CTM results the mixed cluster (2) is likely to include particles emitted from different source types and no single geographical source region seems to dominate for this cluster. The back trajectory analysis and meteorological conditions indicated that the marine origin cluster was correlated with westerly winds and high wind speed; this cluster had high concentrations of Na-containing particles, as expected for sea salt particles.

Small cellular particles are released into the surroundings of cells and are proposed to play an important role in intercellular communication and consequently the responses of microbial com-munities to environmental stressors. We studied the connection between the small cellular parti-cles and the efficiency of three culture series of the microalge Phaeodactylum tricornutum and bac-teria (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols from their growth medium. The microorganism growth rate was determined by flow cytometry, protein profiles were examined by protein gel electrophoresis, cultures and small cellular particle isolates were imaged by scanning electron microscopy, and bisphenols were analyzed using gas chroma-tography coupled with tandem mass spectrometry (GC-MS/MS). Higher growth rates of microal-gae were observed in the co-culture than in the axenic microalgal culture, while the presence of bisphenols neither influenced the morphology of the microalgal cells, protein profiles, nor the small cellular particle isolates. Biotic removal of bisphenols ranged from 0% to 71% and differed among the culture series in a compound-specific manner. However, it remains unclear which mechanisms influenced algal growth and bisphenol removal. Further research on the mechanisms of interspecies communication is needed to advance our understanding of microbial communities at the nano-level.

Conformal radiotherapy is a primary treatment in head and neck cancer, which putative adverse effects depend on relatively low doses of radiation delivered to increased volumes of normal tissues. Systemic effects of such treatment include radiation-induced changes in serum lipid profile, yet dose- and volume-dependence of these changes remain to be established. Here we analyzed levels of choline-containing phospholipids in serum samples collected consecutively during the radiotherapy used as the only treatment modality. The LC-MS approach applied in the study enabled the detection and quantitation of 151 phospholipids, including (lyso)phosphatidylcholines and sphingomyelins. No statistically significant differences were found in the pre-treatment samples from patients with different location and stage of cancer. To compensate for potential differences between schemes of radiotherapy the biologically effective doses were calculated and used in the search of correlations with specific lipid levels. We found that the levels of several phospholipids depended on the maximum dose delivered to the gross tumor volume and total radiation energy absorbed by the patient’s body. Increased doses correlated with increased levels of sphingomyelins and reduced levels of phosphatidylcholines. Noteworthy, serum phospholipid levels were associated mainly with volumes of normal tissues irradiated with relatively low doses (i.e., total accumulated dose 20 Gy), which indicated the importance of such effects on the systemic response of the patient’s organism to IMRT.

Biotin-based proximity labeling approaches, such as BioID, have demonstrated their use for the study of mitochondria proteomes in living cells. The use of genetically engineered BioID cell lines enables the detailed characterization of poorly characterized processes such as mitochondrial co-translational import. In this process, translation is coupled to the translocation of the mitochon-drial proteins, alleviating the energy cost typically associated with the post-translational import relying on chaperone systems. However, the mechanisms are still unclear with only few actors identified but none that have been described in mammals yet. We thus profiled the TOM20 prox-isome using BioID, assuming that some of identified proteins could be molecular actors of the co-translational import in human cells. The obtained results showed a high enrichment of RNA bind-ing proteins close to the TOM complex. However, for the few selected candidates, we could not demonstrate a role in the mitochondrial co-translational import process. Nonetheless, we were able to demonstrate additional uses of our BioID cell line. Indeed, the experimental approach used in this study is thus proposed for the identification of mitochondrial co-translational import effec-tors and for the monitoring of protein entry inside mitochondria with a potential application in the prediction of mitochondrial protein half-life.

Verteporfin, a free base benzoporphyrin derivative monoacid ring A, is a photosensitizing drug for photodynamic therapy (PDT) used in the treatment of the wet form of macular degeneration and activated by red light of 689 nm. Here, we present the first direct study of its photofragmentation channels in the gas-phase, conducted using a laser interfaced mass spectrometer across a broad photoexcitation range from 250-790 nm. The photofragmentation channels are compared with the collision-induced dissociation (CID) products revealing similar dissociation pathways characterized by the loss of the carboxyl and ester groups. Complementary solution-phase photolysis experiments indicate that photobleaching occurs in verteporfin in acetonitrile; a notable conclusion, as photoinduced activity in Verteporfin was not thought to occur in homogenous solvent conditions. These results provide unique new information on the thermal break-down products and photoproducts of this light-triggered drug.

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5–109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86–9.92%. The limit of quantification (LOQ) was set at 8 pg mL–1 for SPA1-H and at 4 ng mL–1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175–0.0515 mg g–1 of Act d 1 and 0.0252–0.0556 mg g–1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.

Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth as they account for a considerable fraction of the total lipids in plants and fungi which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, strawberry) were analyzed and revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0 – h26:0). Interestingly, six GIPCs containing t18:2 were observed in raspberry, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment potentially leading to the identification of new GIPCs. For the first time, automated high‑throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

Abstract: Formation of DNA adducts is a key event for a genotoxic mode of action and its formation is often use as surrogate for mutation and cancer. Interest in DNA adducts are twofold, first, to demonstrate exposure, and second, to link DNA adduct location to subsequent mutations or altered gene regulation. High chemically specific mass spectrometry methods have been established for DNA adduct quantitation and elegant bio-analytic methods utilizing enzymes, various chemistries, and molecular biology methods to visualize the location of DNA adducts. Traditionally, these highly specific methods cannot be combined, and the results are incomparable. Initially developed for single-molecule DNA sequencing, nanopore-type technologies are expected to enable simultaneous quantitation and location of DNA adducts across the genome. We will briefly summarize the current methodologies for state-of-the-art quantitation of DNA adduct levels and mapping of DNA adducts and describe novel single-molecule DNA sequencing technology that is expected to achieve both measures simultaneously. Emerging technologies are expected to soon provide a comprehensive picture of the exposome and identify gene regions susceptible to DNA adduct formation.

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of about 10,000 different soluble and membrane proteins in humans. It involves co- or post-translational targeting of precursor polypeptides to the ER and their subsequent membrane insertion or translocation. So far, three pathways for ER targeting of precursor polypeptides plus four pathways for ER targeting of mRNAs were described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in targeting and, putatively, inserting monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose if this pathway may play a more general role in ER protein targeting, i.e. represents a fourth pathway for ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach, which involves label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells and differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3-clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices and belonging to the secretory pathway were also negatively affected by PEX3-deficiency, which may suggest compromised collagen biogenesis as a hitherto unknown contributor to organ failures in the respective Zellweger patients.

Asphaltenes constitute a heavy fraction of fossil fuels and their characterization is still a very difficult and challenging issue due to their complex and variable composition. Asphaltene components are highly condensed aromatic molecules having some heteroatom and aliphatic functionalities. Their molecular weights distribution span a wide range, from hundreds to millions of units, in dependence on the diagnostic used, leading to speculation about possible occurrence of self-aggregation. In the present work, mass spectrometry, with properly developed mathematical methods, size ex-clusion chromatography and X-ray diffraction analysis have been applied to asphaltenes for giving some further insight on their MW distribution and characteristics. The results here reported give further quantitative support to the experimental data interpretation already reported in previous works.

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in a brand of PAL-labelled milk- and egg-free biscuits. We evaluated the reactivity of children with severe milk and egg allergy, by oral food challenge. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed at immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were found under the limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Our biscuits did not contain allergens of clinical significance. Their milk/egg content is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergenic foods and can be used to avoid unnecessary PAL.

Untargeted metabolomics is a powerful tool for measuring and understanding complex biological chemistries. However, employment, bioinformatics and downstream analysis of mass spectrometry (MS) data can be daunting for inexperienced users. Numerous open-source and free-to-use data processing and analysis tools exist for various untargeted MS approaches, including liquid chro-matography (LC), but choosing the ‘correct’ pipeline isn’t straight-forward. This tutorial, in con-junction with a user-friendly online guide presents a workflow for connecting these tools to process, analyse and annotate various untargeted MS datasets. The workflow is intended to guide explor-atory analysis in order to inform decision-making regarding costly and time-consuming down-stream targeted MS approaches. We provide practical advice concerning experimental design, organisation of data and downstream analysis, and offer details on sharing and storing valuable MS data for posterity. The workflow is editable and modular, allowing flexibility for updated/ changing methodologies and increased clarity and detail as user participation becomes more common. Hence, the authors welcome contributions and improvements to the workflow via the online repository. We believe that this workflow will streamline and condense complex mass-spectrometry approaches into easier, more manageable, analyses thereby generating opportunities for researchers previously discouraged by inaccessible and overly complicated software.

The literature was scoped to help place mass spectrometric (MS) platforms in the spotlight of the life sciences, as a tool of OMICS and OMICS 2.0 technologies. Moreover, today’s appliances are so handy, that they literally place a whole lab on a single chip. Methods of OMICS (and the most recently developed term OMICS 2.0) are tackled, having in mind their aptness for educational purposes, as well as their feasibility. This same topic was highlighted in many reviews that have been periodically published. The author’s initial interest was the forensic application of MS, but since these interests changed, terms such as “biosensors” were also examined, so the review included means of discovery of efficient biomarkers and clinical therapeutic targets. Aside from simply reviewing the research status and the future perspectives, this article tackles terms such as Society 5.0 and OMICS 2.0. However, within the framework of this review, physical chemistry, bioinformatics, or specific software solutions were not specifically studied. Since the topic of generating permanent collections of digital content for retrospective analysis was also reviewed, the need for loads of data – cloud storage was discussed in the context of MS. These large quantities of data are easier to operate with if generated by MS, rather than by any other commercially available assay. In this manner, the review has strengthened an in-depth understanding of the applications of MS.

The European Union's recent decision to renew the authorization for the use of glyphosate until 15 December 2033 stimulates the scientific discussion all around the world regarding its toxicity or otherwise for humans. Glyphosate is a chemical used by millions of tons in the last 50 years worldwide to drying weeds in cultivated fields, greenhouses, roadsides. Concern has been raised in many quarters about the possible presence in the food chain and the consequent adverse effects on health: both aspects that argue in favor of toxicity and those that instead may indicate limited toxicity of glyphosate are here discussed. The great debate that has been generated requires further investigations and field measurements to understand its fate once dispersed in the environment and its concentration in the food chain. Hence the need for validated analytical methods that are available to analysts in the field. In the present review, methods for the analytical determination of glyphosate and its main metabolite, AMPA, are discussed, with a specific focus on chromatographic techniques applied to cereal products. The experimental procedures are explained in any detail, including cleanup, derivatization, and instrumental conditions to give the laboratories involved enough information to proceed with the implementation of this line of analysis. The prevalent chromatographic methods used are LC-MS/MS, GC-MS/SIM, and GC-MS/MS but sufficient indications are also given to those laboratories that wish to use the simpler HPLC-FLD, HPLC-UV, GC-NPD or GC-FPD techniques for screening purposes. Concentrations from literature measured in wheat, corn, barley, rye, oats, soybean, and cereal-based foods are reported together with the regulatory status in various parts of the world and the accumulation mechanism. As for the accumulation in cereals, available data show that glyphosate tends to accumulate more in wholemeal flours than in refined ones, that its concentration in the product strictly depends on the treatment period (the closer it is to the time of harvesting, the higher the concentration) and that in cold climates the herbicide tends to persist in the soil for a long time.

Unmetamorphosed carbonaceous chondrites provide important information concerning the formation and evolution of organic matter, such as amino acids. However, terrestrial contamination remains a valid concern when investigating the organic inventory of meteorites that have fallen to Earth. Accordingly, JAXA’s Hayabusa2 and NASA’s OSIRIS-REx have been launched with the task of returning uncontaminated C-type asteroid material to Earth. The successful Hayabusa2 mission has a very limited sample size (5.4 g). Therefore, many conventional compound specific techniques will struggle to detect amino acids above detection limit with available sample amounts (~several mg to 10’s of mg) being much smaller than those typically used. Here a novel method utilizing ultrahigh performance liquid chromatography-Orbitrap-mass spectrometry is validated and applied to very small meteorite samples, thus providing an approach that can overcome the small sample size constraints of sample return missions. The method is highly sensitive, enabling the detection of amino acids in the carbonaceous chondrites Murchison (2.28 mg) and Orgueil (2.30 mg). Furthermore, quantitation was possible for many of the detected amino acids in Murchison and Orgueil. Using the data presented here, both the amino acid reservoirs of Murchison and Orgueil and the potential application of this method to sample return samples are discussed.

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) of histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis with high-field Fourier transform ion cyclotron resonance MS (FTICR-MS) can detect species missed by bottom-up approaches. We used high-resolution reversed-phase liquid chromatography (RPLC) FTICR-MS, alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. First online RPLC separation sorted histone families then weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative PTM identifications were assigned by matching peptide masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for Histone-intact protein PTM mapping (HiPTMap) and to quantify PTMs on core histones purified from CD8+ T cells directly isolated ex vivo post-influenza infection. Activation significantly reduced PTMs in vivo following influenza infection, histone maps changed as T cells migrated to infections, and T cells responding to secondary heterologous infections had significantly more PTMs enhancing transcriptional activation. Thus, HiPTMap identifies and quantifies PTMs on CD8+ T cell histones and determines their combinations in T cell states.

In recent years, novel food is becoming an emerging trend increasingly more demanding in developed countries. Food proteins from vegetables (pulses, legumes, cereals), fungi, bacteria and insects are being researched to introduce them in meat alternatives, beverages, baked products and others. One of the most complex challenges for introducing novel foods on the market is to assure food safety. New alimentary scenarios drive the detection of novel allergens that need to be identified and quantified with the aim of appropriate labelling. Allergenic reactions are mostly caused by proteins of great abundance in foods, most frequently of small molecular mass, glycosylated, water-soluble and with high stability to proteolysis. The most relevant plant and animal food allergens such as lipid transfer proteins, profilins, seed storage proteins, lactoglobulins, caseins, tropomyosins and parvalbumins from fruits, vegetables, nuts, milk, eggs, shellfish, and fish have been investigated. New methods for massive screening in the search of potential allergens must be developed particularly concerning protein databases and other online tools. Moreover, several bioinformatic tools based on sequence alignment, motif identification or 3-D structure predictions should be implemented as well. Finally, targeted proteomics will become a powerful technology for the quantification of these hazardous proteins. The ultimate objective is to build an effective and resilient surveillance network with this cutting-edge technology.

For more than twenty years chemists use peptidic organocatalysts to transfer acyl groups onto alcohols. The goal was and still is the increase of selectivity towards the substrate of choice. One carefully designed example is a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols. Until now, the intermediate responsible for the acyl transfer of this tetrapeptide has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry. Different mass spectrometric experiments using a nano-ElectroSpray Ionization (ESI) source and tandem MS-techniques allowed the identification of tetrapeptide acylium ions using different acylation reagents. The complexes of trans-cyclohexane-1,2-diols with the tetrapeptide were also detected. Additionally, we were able to detect acylated tetrapeptides in solution using NMR-spectroscopy and monitor the acetylation reaction of a trans-cyclohexane-1,2-diol. These findings are important steps towards the understanding of this organocatalyst and its high enantioselectivity.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respir-atory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking to determin-ing complex host response patterns in differentiated respiratory tract epithelia to understand NiV replication and spread in swine. Here we characterized infection and spread of NiV in differenti-ated primary porcine bronchial epithelial cells (PBEC) cultivated at the air-liquid-interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the api-cal or basal sides. Deep time course proteomics revealed pronounced upregulation of genes re-lated to type I/II- IFN, immunoproteasomal subunits, TAP-mediated peptide transport and MHC I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II-IFN host response with con-version from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.

Breath volatile organic compound analysis is a non-invasive tool for assessing health status; the compositional profile of these compounds in the breath of patients with chronic kidney disease is believed to change with decreasing renal function. We aimed to identify breath volatile organic compounds for recognizing patients with chronic kidney disease. Using thermal desorption-gas chromatography-mass spectrometry, untargeted analysis of breath markers was performed using breath samples of healthy controls (n=18) versus non-dialysis (n=21) and hemodialysis (n=12) patients with chronic kidney disease in this cross-sectional study. A total of 303 volatile organic compounds alongside 12 clinical variables were used to determine the breath volatile organic compound profile. Metabolomic analysis revealed that age, systolic blood pressure, and fifty-eight breath volatile organic compounds differed significantly between the chronic kidney disease group (non-dialysis + hemodialysis) and healthy controls. Thirty-six volatile organic compounds and two clinical variables that showed significant associations with chronic kidney disease in the univariate analysis were further analyzed. Different spectras of breath volatile organic compounds between the control and chronic kidney disease groups were obtained. Multivariate model incorporating age, 2-methyl-pentane, and cyclohexanone showed high performance (accuracy, 86%) in identifying patients with chronic kidney disease, with odds ratios (95% confidence interval) P-values of 0.18 (0.07-2.49) 0.013; 2.10 (0.94-2.24) 0.025; and 2.31 (0.88–2.64) 0.008, respectively. Hence, renal dysfunction-associated characteristic profiles of breath volatile organic substances can be used as non-invasive markers to screen for chronic kidney disease.

The metalloporphyrin heme acts as the oxygen-complexing prosthetic group of hemoglobin in blood. Heme has been noted to survive for many millions of years in fossils. Here we investigate its stability and degra-dation under various conditions expected to occur during fossilization. Oxidative, reductive, aerobic, and anaerobic conditions were studied at neutral and alkaline pH values. Elevated temperatures were applied to accelerate degradation. High-performance liquid chromatography coupled to tandem mass spectrome-try (HPLC-MS/MS) identified four main degradation products. The vinyl residues are oxidized to formyl and further to carboxylate groups. In the presence of air or H2O2, cleavage of the tetrapyrrole ring occurs, and hematinic acid is formed. The highest stability of heme was observed under anaerobic reductive condi-tions (half-life 9.5 days), while the lowest stability was found in the presence of H2O2 (half-life 1 min). We confirmed that the iron cation plays a crucial role in degradation, since protoporphyrin IX, lacking iron, re-mained significantly more stable. Under anaerobic, reductive conditions, the above-mentioned degradation products were not observed, suggesting a different degradation pathway. To our knowledge, this is the first molecular taphonomy study on heme, which will be useful for understanding its fate during fossiliza-tion.

Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography dynamic multiple reaction monitoring targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of Chronic Myeloid Leukaemia (CML), we found that intra- and extra-cellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), and extracellular Prostaglandin F_2α (PGF_2α), 5-Hydroxyeicosatetraenoic acid (5-HETE), Palmitic acid (PA, C16:0) and Stearic acid (SA, C18:0) were altered in response to immunomodulation by type I Interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism with potential relevance in the context of cancer inflammation and CML.