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Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are major lipids in plant and fungal plasma membranes and play an important role in stress adaption. However, their analysis remains challenging due to the multiple steps involved in their extraction and purification prior to mass spectrometry analysis. To address this challenge, we report here a novel simplified method to identify GIPCs from Aspergillus fumigatus using the new Bruker MBT lipid Xtract assay. A. fumigatus reference strains and clinical isolates were cultured, harvested, heat-inactivated and suspended in double-distilled water. A fraction of this fungal preparation was then dried in a microtube, mixed with an MBT lipid Xtract matrix (Bruker Daltonik, Germany) and loaded onto a MALDI target plate. Analysis was performed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were scanned from m / z 700 to m / z 2 000. MALDI-TOF MS analysis of cultured fungi showed a clear signature of GIPCs in Aspergillus fumigatus reference strains and clinical isolates. Here, we have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with the MBT lipid Xtract is able to detect Aspergillus fumigatus GIPCs.

Introduction: The human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease., Objectives: Here, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics., Methods: Comparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data., Results: Glycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors., Conclusion: Our work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Contributing authors Pierre-Olivier Schmit, Stuart Pengelley, and Kristina Marx are employees of Bruker Daltonics which is the manufacturer of some of the hardware and software that were used in this work., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Background: Protein analysis using matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometry (MALDI-TOF MS) represents a promising tool for entomological surveillance. In this study we tested the discriminative power of this tool for measuring species and blood meal source of main Afrotropical malaria vectors on the Kenyan coast., Methods: Mosquito collections were conducted along the coastal region of Kenya. MALDI-TOF MS spectra were obtained from each individual mosquito's cephalothorax as well as the abdomens of blood-engorged mosquitoes. The same mosquitoes were also processed using gold standard tests: polymerase chain reaction (PCR) for species identification and enzyme linked immunosorbent assay (ELISA) for blood meal source identification., Results: Of the 2,332 mosquitoes subjected to MALDI-TOF MS, 85% (1,971/2,332) were considered for database creation and validation. There was an overall accuracy of 97.5% in the identification of members of the An. gambiae ( An. gambiae , 100%; An. arabiensis , 91.9%; An. merus , 97.5%; and An. quadriannulatus , 90.2%) and An. funestus ( An. funestus , 94.2%; An. rivulorum , 99.4%; and An. leesoni , 94.1%) complexes. Furthermore, MALDI-TOF MS also provided accurate (94.5% accuracy) identification of blood host sources across all mosquito species., Conclusions: This study provides further evidence of the discriminative power of MALDI-TOF MS to identify sibling species and blood meal source of Afrotropical malaria vectors, further supporting its utility in entomological surveillance. The low cost per sample (<0.2USD) and high throughput nature of the method represents a cost-effective alternative to molecular methods and could enable programs to increase the number of samples analysed and therefore improve the data generated from surveillance activities., Competing Interests: Competing interests: Bruker did not fund or participate in study design or analysis; however, the authors declare that they have obtained technical support for quality control purposes. Bruker provided guidance/assistance in database creation and validation and Mumin Eminov is affiliated with Bruker Daltonik GmbH. No other competing interests were disclosed., (Copyright: © 2024 Karisa J et al.)

Infrared ion spectroscopy (IRIS) can be used to identify molecular structures detected in mass spectrometry (MS) experiments and has potential applications in a wide range of analytical fields. However, MS-based approaches are often combined with orthogonal separation techniques, in many cases liquid chromatography (LC). The direct coupling of LC and IRIS is challenging due to the mismatching timescales of the two technologies: an IRIS experiment typically takes several minutes, whereas an LC fraction typically elutes in several seconds. To resolve this discrepancy, we present a heartcutting LC-IRIS approach using a setup consisting of two switching valves and two sample loops as an alternative to direct online LC-IRIS coupling. We show that this automated setup enables us to record multiple IR spectra for two LC-features from a single injection without degrading the LC-separation performance. We demonstrate the setup for application in drug metabolism research by recording six m/z-selective IR spectra for two drug metabolites from a single 2 μL sample of cell incubation extract. Additionally, we measure the IR spectra of two closely eluting diastereomeric biomarkers for the inborn error of metabolism pyridoxine-dependent epilepsy (PDE-ALDH7A1), which shows that the heartcutting LC-IRIS setup has good sensitivity (requiring ∼μL injections of ∼μM samples) and that the separation between closely eluting isomers is maintained. We envision applications in a range of research fields, where the identification of molecular structures detected by LC-MS is required.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories., Competing Interests: Conflict of interest F. B., M. L., and G. K. are employees of Bruker Daltonik GmbH, manufacturer of the timsTOF Pro, and thus declare no competing interests. All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Honey is a highly consumed commodity due to its potential health benefits upon certain consumption, resulting in a high market price. This fact indicates the need to protect honey from fraudulent acts by delivering comprehensive analytical methodologies. In this study, targeted, suspect and non-targeted metabolomic workflows were applied to identify botanical origin markers of Greek honey. Blossom honey samples ( n = 62) and the unifloral fir ( n = 10), oak ( n = 24), pine ( n = 39) and thyme ( n = 34) honeys were analyzed using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) system. Several potential authenticity markers were revealed from the application of different metabolomic workflows. In detail, based on quantitative targeted analysis, three blossom honey markers were found, namely, galangin, pinocembrin and chrysin, while gallic acid concentration was found to be significantly higher in oak honey. Using suspect screening workflow, 12 additional bioactive compounds were identified and semi-quantified, achieving comprehensive metabolomic honey characterization. Lastly, by combining non-targeted screening with advanced chemometrics, it was possible to discriminate thyme from blossom honey and develop binary discriminatory models with high predictive power. In conclusion, a holistic approach to assessing the botanical origin of Greek honey is presented, highlighting the complementarity of the three applied metabolomic approaches.

Astronauts experience dramatic loss of muscle mass, decreased strength, and insulin resistance, despite performing daily intense physical exercise that would lead to muscle growth on Earth. Partially mimicking spaceflight, prolonged bed rest causes muscle atrophy, loss of force, and glucose intolerance. To unravel the underlying mechanisms, we employed highly sensitive single fiber proteomics to detail the molecular remodeling caused by unloading and inactivity during bed rest and changes of the muscle proteome of astronauts before and after a mission on the International Space Station. Muscle focal adhesions, involved in fiber-matrix interaction and insulin receptor stabilization, are prominently downregulated in both bed rest and spaceflight and restored upon reloading. Pathways of antioxidant response increased strongly in slow but not in fast muscle fibers. Unloading alone upregulated markers of neuromuscular damage and the pathway controlling EIF5A hypusination. These proteomic signatures of mechanical unloading in muscle fiber subtypes contribute to disentangle the effect of microgravity from the pleiotropic challenges of spaceflight., (© The Author(s) 2022. Published by Oxford University Press on behalf of the National Academy of Sciences.)

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila ( Lp ) is the species most frequently responsible for a severe pneumonia known as Legionnaires' disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. With regard to Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approaches for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating from a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at the serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates ( n = 133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of the instrument's classifier, was tested by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). All isolates were classified as follows: 12/133 (9.0%) as Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 that returned as Lp Sg1 was observed, and 4/133 (3.0%) isolates were not classified. An accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared with agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs, especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies., Competing Interests: MC was employed by Bruker Daltonic GmbH (the manufacturer of IR-Biotyper®). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pascale, Bisognin, Mazzotta, Girolamini, Marino, Dal Monte, Cordovana, Scaturro, Ricci and Cristino.)

Tuberculosis represents one of the ten most common courses of death worldwide and the emergence of multidrug-resistant M. tuberculosis makes the discovery of novel anti-tuberculosis active structures an urgent priority. Here, we show that (+)-floyocidin B representing the first example of a novel dihydroisoquinoline class of fungus-derived natural products, displays promising antitubercular hit properties. (+)-Floyocidin B was identified by activity-guided extract screening and its structure was unambiguously determined by total synthesis. The absolute configuration was deduced from a key synthesis intermediate by single crystal X-ray diffraction analysis. A hit series was generated by the isolation of further natural congeners and the synthesis of analogs of (+)-floyocidin B. Extensive biological and physicochemical profiling of this series revealed first structure-activity relationships and set the basis for further optimization and development of this novel antitubercular scaffold., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease., (© 2022 The Authors Published under the terms of the CC BY 4.0 license.)

The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the virus life cycle and is supposed to be a potential target for the treatment of coronaviral infection. Traditional Chinese medicines (TCMs) have played an impressive role in the treatment of COVID-19 in China. The effectiveness of TCM formulations prompts scientists to take continuous effort on searching for bioactive small molecules from the ancient resources. Herein, we developed a native mass spectrometry-based affinity-selection method for rapid screening of active small molecules from crude herbal extracts applied for COVID-19 therapy. Six common herbs named Lonicera japonica, Scutellaria baicalensis, Forsythia suspensa, Glycyrrhiza uralensis, Cirsium japonicum, and Andrographis paniculata were investigated. After preliminary separation of the crude extracts, the fractions were incubated with 3CLpro. A native MS-based affinity screening assay was then conducted to search for the protein-ligand complexes. A UHPLC-Q/TOF-MS with UNIFI data acquisition and data processing software was applied to identify the hit compounds. Standard compounds were used to verify the outcomes. Among the 16 hits, three flavonoids, baicalein, scutellarein and ganhuangenin, were identified as potential noncovalent inhibitors against 3CLpro with IC 50 values of 0.94, 3.02, and 0.84 μM, respectively. Their binding affinities were further characterized by native MS, with K d values being 1.43, 3.85, and 1.09 μM, respectively. Overall, we established an efficient native MS-based strategy for discovering 3CLpro ligands from crude mixtures, which supplies a potential strategy of small molecule lead discovery from TCMs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mass spectrometry (MS)-based quantitative proteomic methods have become some of the major tools for protein biomarker discovery and validation. The recently developed parallel reaction monitoring-parallel accumulation-serial fragmentation (prm-PASEF) approach on a Bruker timsTOF Pro mass spectrometer allows the addition of ion mobility as a new dimension to LC-MS-based proteomics and increases proteome coverage at a reduced analysis time. In this study, a prm-PASEF approach was used for the multiplexed absolute quantitation of proteins in human plasma using isotope-labeled peptide standards for 125 plasma proteins, over a broad (10 4 -10 6 ) dynamic range. Optimization of LC and MS parameters, such as accumulation time and collision energy, resulted in improved sensitivity for more than half of the targets (73 out of 125 peptides) by increasing the signal-to-noise ratio by a factor of up to 10. Overall, 41 peptides showed up to a 2-fold increase in sensitivity, 25 peptides showed up to a 5-fold increase in sensitivity, and 7 peptides showed up to a 10-fold increase in sensitivity. Implementation of the prm-PASEF method allowed absolute protein quantitation (down to 1.13 fmol) in human plasma samples. A comparison of the concentration values of plasma proteins determined by MRM on a QTRAP instrument and by prm-PASEF on a timsTOF Pro revealed an excellent correlation ( R 2 = 0.97) with a slope of close to 1 (0.99), demonstrating that prm-PASEF is well suited for "absolute" quantitative proteomics.

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Oncogenic drivers such as mutated EGFR are the preferred targets in modern drug development. However, restoring the lost function of tumor suppressor proteins could also be a valid approach to combatting cancer. ITIH5 has been revealed as a potent metastasis suppressor in both breast and pancreatic cancer. Here, we show that ITIH5 overexpression in MDA-MB-231 breast cancer cells can also locally suppress tumor growth by 85%, when transplanted into the mammary fat pad of nude mice. For a potential drug development approach, we further aimed to define downsized ITIH5 polypeptides that still are capable of mediating growth inhibitory effects. By cloning truncated and His-tagged ITIH5 fragments, we synthesized two recombinant N -terminal polypeptides (ITIH5 681aa and ITIH5 161aa ), both covering the ITI heavy chain specific "vault protein inter-alpha-trypsin" (VIT) domain. Truncated ITIH5 variants caused dose-dependent cell growth inhibition by up to 50% when applied to various cancer cell lines (e.g., MDA-MB-231, SCaBER, A549) reflecting breast, bladder and lung cancer in vitro. Thus, our data suggest the substantial role of the ITIH5-specific VIT domain in ITIH5-mediated suppression of tumor cell proliferation. As extracellularly administered ITIH5 peptides mimic the growth-inhibitory effects of the full-length ITIH5 tumor suppressor protein, they may constitute the basis for developing anticancer drugs in the future.

Highlighter inks were analyzed by means of soft Desorption/Ionization induced by Neutral SO 2 clusters (DINeC) in combination with mass spectrometry (MS). The dye molecules of the different inks were directly desorbed from dots of ink drawn on arbitrary substrates. Fragmentation free spectra were observed and the dyes used in the dye mixtures of the different highlighter inks were unambiguously identified. The soft nature of cluster-induced desorption was used to investigate the decomposition of the dye molecules induced by either heat or UV-light. The two processes lead to different decomposition products which are clearly distinguished in the DINeC spectra. The two different degradation processes can thus be discriminated using DINeC-MS.

Trapped ion mobility spectrometry (TIMS) is a promising technique for the separation of isomers based on their mobility. In the present work, TIMS coupled to liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) was applied as a comprehensive analytical platform to address authenticity challenges, focusing on extra virgin olive oil (EVOO). Isomers detected in EVOO's phenolic fraction, classified into secoiridoids group, were successfully separated. Thanks to parallel accumulation serial fragmentation (PASEF) acquisition mode, high-quality spectra were obtained, facilitating identification. Moreover, a four-dimensional (4D) untargeted metabolomics approach was implemented to evaluate EVOO's global profile in cases of both variety and geographical origin discrimination. Potential authenticity markers, attributed to isomers, were successfully identified through the proposed workflow that incorporates ion mobility information along with LC-HRMS analytical evidence (i.e., mass accuracy, retention time, isotopic pattern, MS/MS fragmentation). Our study establishes LC-TIMS-HRMS in food authenticity and highlights mobility-enhanced metabolomics in four dimensions.

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA-hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA's bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies-BoxCar acquisition and trapped ion mobility spectrometry-both lead to deep and accurate proteome quantification., (© 2021. The Author(s).)

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plant genebanks constitute a key resource for breeding to ensure crop yield under changing environmental conditions. Because of their roles in a range of stress responses, phenylpropanoids are promising targets. Phenylpropanoids comprise a wide array of metabolites; however, studies regarding their diversity and the underlying genes are still limited for cereals. The assessment of barley diversity via genotyping-by-sequencing is in rapid progress. Exploring these resources by integrating genetic association studies to in-depth metabolomic profiling provides a valuable opportunity to study barley phenylpropanoid metabolism; but poses a challenge by demanding large-scale approaches. Here, we report an LC-PDA-MS workflow for barley high-throughput metabotyping. Without prior construction of a species-specific library, this method produced phenylpropanoid-enriched metabotypes with which the abundance of putative metabolic features was assessed across hundreds of samples in a single-processed data matrix. The robustness of the analytical performance was tested using a standard mix and extracts from two selected cultivars: Scarlett and Barke. The large-scale analysis of barley extracts showed (1) that barley flag leaf profiles were dominated by glycosylation derivatives of isovitexin, isoorientin, and isoscoparin; (2) proved the workflow's capability to discriminate within genotypes; (3) highlighted the role of glycosylation in barley phenylpropanoid diversity. Using the barley S42IL mapping population, the workflow proved useful for metabolic quantitative trait loci purposes. The protocol can be readily applied not only to explore the barley phenylpropanoid diversity represented in genebanks but also to study species whose profiles differ from those of cereals: the crop Helianthus annuus (sunflower) and the model plant Arabidopsis thaliana., (© 2021 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum , it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy., Competing Interests: MP and FD are employees of Probiotical Research S.r.L., Novara, Italy. MC is employee of Bruker Daltonik, Bremen, Germany. FF and IC are employees of Microbion S.r.L., San Giovanni Lupatoto, Verona, Italy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Deidda, Bozzi Cionci, Cordovana, Campedelli, Fracchetti, Di Gioia, Ambretti and Pane.)

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240-247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains ( n =14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains ( n =6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains., Competing Interests: LD, AF, and GL-M are co-inventors of the MALDIxin test for which a patent has been filed by Imperial Innovations (WO2018158573). MK is employee of Bruker, the manufacturer of the MALDI-TOF MS used in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jeannot, Hagart, Dortet, Kostrzewa, Filloux, Plesiat and Larrouy-Maumus.)

The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m / z , isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate., (© 2021. The Author(s).)

Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age-related polyethism characterized by age-related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age-related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants' central nervous system combined with brain extract analysis by Q-Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide-, neuropeptide-like, and protein hormone prepropeptide genes, including a novel neuropeptide-like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage-specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Over the past seven years Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Profiling (MALDI MSP) and Imaging (MALDI MSI) have proven to be feasible tools for the detection of blood and its provenance in stains and fingermarks. However, whilst this capability as a confirmatory test addresses the primary questions at the scene of a violent crime, additional intelligence recoverable from blood can also prove important for investigations. A DNA profile is the most obvious and important example of such intelligence; however, it is not always suitable for identification purposes, depending on quantity, age and environmental conditions. Proteins are much more stable and determining the presence of haemoglobin variants in blood recovered at a crime scene may provide associative and possibly corroborating evidence on the presence of an individual at a particular location. This evidence gains more incriminatory value, the lower the incidence of the variant in a certain geographical area or population and may contribute to narrowing down the pool of suspects. In this study, a MALDI based mass spectrometric method has been developed and tested on six haemoglobin variants for their fast and reliable identification and mapping in blood fingermarks.

A series of aryl N -[ω-(6-fluoroindol-1-yl)alkyl]carbamates with alkyl spacers of varying lengths between the indole and the carbamate group and with differently substituted aryl moieties at the carbamate oxygen were synthesized and tested for inhibition of the pharmacologically interesting serine hydrolases fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), butyrylcholinesterase (BuChE), and acetylcholinesterase (AChE). Furthermore, the chemical stability in an aqueous solution and the metabolic stability toward esterases in porcine liver homogenate and porcine blood plasma were determined. While most of the synthesized derivatives were potent inhibitors of FAAH, a considerable inhibition of MAGL and BuChE was elicited only by compounds with a high carbamate reactivity, as evidenced by a significant hydrolysis of these compounds in an aqueous solution. However, the high inhibitory potency of some compounds toward MAGL and BuChE, especially that of the ortho -carboxyphenyl derivative 37 , could not be explained by chemical reactivity alone. Several of the carbamates studied possessed varying degrees of stability toward esterases from liver and blood plasma. In some cases, marked inactivation by the pseudo-esterase activity of plasma albumin was observed. Mass spectrometric studies showed that such carbamates formed covalent bonds with albumin at several sites., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A . fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A . fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A . fumigatus . This review paper comprehensively discusses the current clinical challenges caused by A . fumigatus and provides insights on how to address them., (© 2021 Westerdijk Fungal Biodiversity Institute. Production and hosting by ELSEVIER B.V.)

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N - and O -glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O -glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S . Typhi, 23 S . Paratyphi A, 23 S . Paratyphi B, and 7 S . Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella . Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes.