Mass Spectrometry Research Articles

Inhibition of diacylglycerol lipase α induced blood–brain barrier breach in female Sprague–Dawley rats

AbstractThe endocannabinoid system's significance in maintaining blood–brain barrier (BBB) integrity under physiological and pathological conditions is suggested by several reports, but the underlying molecular mechanisms are not well understood. In this paper, we investigated the effects of depletion of 2‐arachidonoylglycerol (2‐AG), one of the main endocannabinoids in the central nervous system, on BBB integrity using pharmacological tools. Female Sprague–Dawley rats were injected with the diacylglycerol lipase α (DAGLα) inhibitor LEI‐106 (40 mg/kg, i.p.), followed by assessment of BBB integrity via in situ brain perfusion. Liquid chromatography–mass spectrometry, western immunoblotting, light transmittance experiments and pressure myography were also used to further examine the results of DAGLα blockade on the BBB and vascular reactivity. We found that DAGLα inhibition caused BBB opening in cortical brain areas, manifesting as increased sucrose transport measured by in situ brain perfusion. This was accompanied by reduced levels of 2‐AG and decreased detection of the tight junction protein zonula occludens‐1 (ZO‐1). The protein level in cortical areas of neuronal PAS domain protein 4 (NPAS4), encoded by an activity‐dependent immediate early gene, was increased without the presence of cortical spreading depression after LEI‐106 administration. We also observed a significant increase in pressure‐induced constriction within the parenchymal microcirculation after inhibition of DAGLα, possibly altering shear stress in the microcirculation. These results support the role of endogenous 2‐AG in maintaining normal tight junction function. This improved understanding of the molecular mechanisms of endocannabinoid system function at the neurovascular unit can help to unlock the therapeutic potentials of cannabinoids in central nervous system disorders associated with BBB dysfunction. imageKey points The administration of the diacylglycerol lipase α (DAGLα) inhibitor LEI‐106 (40 mg/kg, i.p.) induced blood–brain barrier (BBB) opening of cortical brain areas in female Sprague–Dawley rats. This BBB disruption was accompanied by reduced levels of 2‐arachidonoylglycerol (2‐AG) and decreased detection of the tight junction protein zonula occludens‐1 (ZO‐1). The protein level in cortical areas of neuronal PAS domain protein 4 (NPAS4), encoded by an activity‐dependent immediate early gene, was increased without the presence of cortical spreading depression after LEI‐106 administration. A significant increase in pressure‐induced constriction within the parenchymal microcirculation was also observed after inhibition of DAGLα, possibly altering shear stress. These results support the role of endogenous 2‐AG in maintaining normal tight junction function.

Urinary odor molecules in the Otassha Study can distinguish patients with sarcopenia: A pilot study

AimTo identify sarcopenia markers in urinary odor.MethodsWe performed solid‐phase microextraction from the headspace and gas chromatography–mass spectrometry analysis of urinary volatile organic compounds (VOCs) in 71 healthy individuals and 68 patients diagnosed with sarcopenia according to the Asian Working Group on Sarcopenia 2019 criteria. The mass‐to‐charge ratios (m/z) of 10 VOCs with a significant difference in the total ion chromatogram of 220 VOCs detected in this study were compared by U‐test. To calculate the predictive values for sarcopenia, binomial logistic regression analyses were conducted with sarcopenia (0, 1) as the dependent variable and the m/z values of each of the 10 VOCs and all 10 VOCs as independent variables. Receiver operating characteristic (ROC) curves for predictive values were generated to evaluate diagnostic accuracy. The correlations between the predictive value and handgrip strength, usual gait speed, and skeletal muscle mass were assessed using Pearson's r.ResultsWe identified 10 VOCs (p‐xylene, 1‐butanol, d‐limonene, nonanal, pyrrole, γ‐butyrolactone, texanol isomer, octanoic acid, nonanoic acid, and diisobutyl phthalate) as candidate biomarkers in urine. The ROC curve analysis showed high diagnostic accuracy of the predictive values of the 10 VOCs for sarcopenia (area under the curve = 0.866, 95% confidence interval: 0.829–0.942; sensitivity, 80.9%; specificity, 81.7%). Additionally, the predictive values significantly correlated with handgrip strength (male: r = −0.505, P < 0.0001; female: r = −0.568, P < 0.0001).ConclusionsThis study identified 10 urinary VOCs as possible non‐invasive biomarkers for sarcopenia, offering insights into its onset mechanism and potential therapeutic targets. Geriatr Gerontol Int 2025; ••: ••–••.

Substrate stiffness dictates unique doxorubicin-induced senescence-associated secretory phenotypes and transcriptomic signatures in human pulmonary fibroblasts.

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-β-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2kPa), fibrotic (50kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21 + cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

New Numerical Inversion Method to Improve the Spatial Accuracy of Elemental Imaging for LA-ICP-MS.

The elemental imaging of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) provides spatial information on elements and therefore can further investigate the growth or evolution processes of an analyte. However, the accurate determination of spatial information is limited by the decoupling between the elemental distribution and mass spectrometry signals. This phenomenon, which is more distinct when high-diffusion ablation cells are used, arises from the overlap of ablation and the transport dispersion of aerosols. Reconstruction of the elemental distribution using mathematical algorithms is an effective approach to addressing this decoupling. This study establishes a comprehensive numerical inversion method that targets the independent events of aerosol diffusion and overlapping ablation in LA-ICP-MS. A new signal fitting model and deconvolution algorithm are employed to mitigate the effects of aerosol diffusion. Moreover, boundary identification and restoration algorithms are developed to resolve the challenges posed by blurring of the phase boundary when the laser beam scans over the two-phase interface. By employing the presented numerical method, the shape of fine materials can be measured with an accuracy of 10%, which is an ∼18-fold improvement compared to the raw data. The new algorithm is expected to enhance the accuracy of the spatial distribution in LA-ICP-MS elemental imaging, particularly for traditional high-diffusion ablation cells, which are still in use in microanalytical laboratories on a global scale. Additionally, the numerical inversion method presented here can be applied across various fields of science to improve the quality of element imaging.

B-doped nano-hydroxyapatite facilitates proliferation and differentiation of osteoblasts

PurposeWe aimed to explore the mechanism by which Boron-doped nano-hydroxyapatite (B-nHAp) facilitates the proliferation and differentiation of osteoblasts through controlled release of B.MethodsB-nHAp characterization was accomplished by means of X-ray diffraction, scanning electron microscopy, inductively coupled plasma mass spectrometry, and transmission electron microscopy. Human bone marrow mesenchymal stem cells (hBMSCs) were subjected to flow cytometry, alizarin red S staining, and cell counting kit-8 assay for proliferation and differentiation determination. Western blotting for protein expression detection together with real-time quantitative polymerase chain reaction for mRNA expression measurement were carried out on those related to hBMSC proliferation and differentiation. Immunofluorescence staining was conducted to determine the activity of the Wnt/β-catenin signaling pathway.ResultsB-nHAp particles had structured configuration and uniform size, and a typical nHAp crystal structure. The B content in B-nHAp was in line with expectation. hBMSCs displayed stemness. B-nHAp significantly facilitated the proliferation of hBMSCs, and significantly more mineralized nodules formed in the B-nHAp group. B-nHAp significantly upregulated the expressions of marker molecules related to hBMSC proliferation and differentiation. B-nHAp boosted the activity of the Wnt/β-catenin signaling pathway.ConclusionB-nHAp modulates the Wnt/β-catenin signaling pathway to significantly enhance the proliferative and differential abilities of osteoblasts, potentially as an efficient material for bone repair.

Structural basis for the interaction between the Drosophila RTK Sevenless (dROS1) and the GPCR BOSS

Sevenless, the Drosophila homologue of ROS1 (University of Rochester Sarcoma) (herein, dROS1) is a receptor tyrosine kinase (RTK) essential for the differentiation of Drosophila R7 photoreceptor cells. Activation of dROS1 is mediated by binding to the extracellular region (ECR) of the GPCR (G protein coupled receptor) BOSS (Bride Of Sevenless) on adjacent cells. Activation of dROS1 by BOSS leads to subsequent downstream signaling pathways including SOS (Son of Sevenless). However, the physical basis for how dROS1 interacts with BOSS has long remained unknown. Here we provide a cryo-EM structure of dROS1’s extracellular region, which mediates ligand binding. We show that the extracellular region of dROS1 adopts a folded-over conformation stabilized by an N-terminal domain comprised of two disulfide stapled helical hairpins. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS using hydrogen-deuterium exchange mass spectrometry (HDX-MS). This includes beta-strands in dROS1’s third Fibronectin type III (FNIII) domain and a C-terminal peptide in BOSS’ ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and beta-strand augmentation between these regions. Our findings provide a fundamental understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1.

Profiles of testosterone and pre-androgens and sexual function in premenopausal women

Abstract Background There is inconsistent evidence as to the role of testosterone and pre-androgens in premenopausal female sexual function, and reported associations between blood concentrations of these hormones and female sexual function vary in strength. Aim To examine the patterns of testosterone and pre-androgen concentrations and variations in sexual function in premenopausal eumenorrheic women. Methods This was a secondary analysis of a sample of 588 premenopausal eumenorrheic women from the Grollo-Ruzzene Foundation Young Women’s Health Study. Socio-demographics, health information, and questionnaire data were collected using online surveys. Eligible women were invited to provide a blood sample. We ran latent profile analysis (LPA) and subsequent analyses in R using RStudio. Outcomes Indicator variables in the LPA included sexual arousal and desire domains of the Profile of Female Sexual Function and testosterone, dehydroepiandrosterone (DHEA), and androstenedione, measured by liquid chromatography–tandem mass spectrometry. Results Analyses resulted in a pattern of 3 latent classes. Classes reporting relatively lower and higher sexual arousal (LPA-derived means and 95% CIs: -0.79 [-1.24; -0.34] and 0.62 [0.51; 0.72]) did not differ significantly in sex steroid concentrations (testosterone: -0.21 [-0.38; -0.03] and -0.33 [-0.47; -0.20]; DHEA: -0.47 [-0.57; -0.37] and -0.26 [-0.39; -0.13]; androstenedione: -0.36 [-0.50; -0.22] and -0.39 [-0.49; -0.29]), while the class reporting relatively medium arousal (-0.11 [-0.31; 0.08]) showed the highest testosterone, DHEA, and androstenedione concentrations (testosterone: 0.8 [0.60; 1.01]; DHEA: 0.99 [0.76; 1.23]; androstenedione: 1.08 [0.88; 1.29]). There were no significant differences in sexual desire between classes (-0.08 [-0.23; 0.06]; 0.00 [-0.13; 0.14]; 0.10 [-0.09; 0.30]) differing significantly in sex steroid concentrations (-0.69 [-0.80; -0.58], -0.04 [-0.15; 0.07], 0.94 [0.71; 1.16] for testosterone) nor associations between the sex steroid concentrations and degrees of sexual desire. Clinical Implications These findings cast further doubt on the utility of measuring sex steroids for diagnosing female sexual dysfunction in premenopausal eumenorrheic women, even when considered in combination. Strengths and Limitations We analyzed a large community sample and controlled for potentially biasing factors. We analyzed sex steroid concentrations determined with gold-standard methodology. Excluding women with early menopause and menstrual dysfunction might have resulted in finding 3, rather than more, latent classes. Conclusion Testosterone and pre-androgen profiles do not clearly identify premenopausal eumenorrheic women with low sexual arousal and desire.

Ultrafast Dissociation Dynamics of the Sensitive Explosive Ethylene Glycol Dinitrate.

Ethylene glycol dinitrate (EGDN) is a nitrate ester explosive widely used in military ordnance and missile systems. This study investigates the decomposition dynamics of the EGDN cation using a comprehensive approach that combines femtosecond time-resolved mass spectrometry (FTRMS) experiments with ab initio electronic structure and molecular dynamics computations. We identify three distinct dissociation time scales for the metastable EGDN cation of approximately 40-60 fs, 340-450 fs, and >2 ps. The observed dissociation time scales are rationalized by electronic and geometric relaxation of multiple EGDN conformers. These insights are crucial for advancing knowledge of the initial molecular decomposition processes that are central to the detonation physics of nitrate esters, which can lead to improving safety protocols and optimizing the performance of nitrate ester explosives in various applications.

Optimised determination of arsenic and lead in tea samples using microwave-assisted digestion and ICP-MS: method validation and health risk assessment

ABSTRACT Heavy metal contamination in tea poses significant risks to consumer health, necessitating the development of accurate and efficient analytical methods for routine monitoring. This study aimed to optimise and validate a robust process for the simultaneous determination of arsenic (As) and lead (Pb) in dried tea samples using inductively coupled plasma mass spectrometry (ICP-MS) following microwave-assisted digestion. These toxic elements were selected due to their known health risks and their frequent detection in tea. The method was systematically optimised for digestion parameters and validated following EURACHEM guidelines to ensure accuracy and precision. Key validation metrics included linearity (R2 ≥0.9999), limits of quantification (LOQ) of 0.25 µg/kg for As and 1.5 µg/kg for Pb, sufficient for regulatory compliance. The method demonstrated excellent mean recoveries (75%–89%) and relative standard deviations (RSDs) below 15% across all fortification levels. Precision and accuracy were further verified through proficiency testing, yielding acceptable z-scores for both analytes. Application of the validated method to tea samples collected from Egypt revealed arsenic concentrations ranging from 0.018 ± 0.004 to 0.280 ± 0.070 mg/kg and lead concentrations from 0.083 ± 0.021 to 1.630 ± 0.407 mg/kg. Although detection rates were elevated, the calculated target hazard quotient (THQ) values for As and Pb were below the threshold of 1, suggesting no significant non-carcinogenic health risks associated with tea consumption. This study provides a reliable, cost-effective analytical framework for monitoring heavy metal contamination in tea products, supporting consumer safety and public health initiatives.

The First Large Identification of 3ANX and NX Producing Isolates of Fusarium graminearum in Manitoba, Western Canada

Fusarium head blight, caused by Fusarium graminearum, continues to be one of the most important and devastating fungal diseases on cereal grains including wheat, barley, and oat crops. F. graminearum produces toxic secondary metabolites that include trichothecene type A and type B mycotoxins. There are many variants of these toxins that are produced, and in the early 2010s, a novel type A trichothecene mycotoxin known as 3ANX (7-α hydroxy,15-deacetylcalonectrin) and its deacetylated product NX (7-α hydroxy, 3,15-dideacetylcalonectrin) were identified in Minnesota, USA. In the current study, a total of 31,500 wheat spikes over a period of 6 years (2015–2020) within Manitoba, Canada, were screened for the F. graminearum pathogen, which accounted for 72.8% (2015), 98.3% (2016), 71.9% (2017), 74.4% (2018), 92.6% (2019), and 66.1% (2020) of isolations. A total of 303 F. graminearum isolates, confirmed through sequencing of the ribosomal intergenic spacer, were further investigated for variation in the gene Tri1, which was previously associated with the production of the NX toxin, as well as the accumulation of mycotoxins. A subset of these isolates, consisting of 73 isolates, which tested positive or negative for the NX-Tri1-F/R assay in this study, were cultured in vitro using rice media. Mycotoxins were quantified in these samples using mass spectrometry. Using the same rice culture, genomic DNA was isolated, and the Tri1 coding sequence along with its flanking regions (upstream and downstream of the Tri1 gene) was amplified and sequenced. Deoxynivalenol (DON) accumulated in 96% of the cultures from these isolates, while 3-acetyl deoxynivalenol (3ADON) and 3ANX mycotoxins accumulated in 66% and 63%, respectively. Nivalenol, 15-acetyl deoxynivalenol, and NX mycotoxins were detected in 62%, 36%, and 19% of samples, respectively. A significant correlation was observed between 3ADON and 3ANX (r2 = 0.87), as well as between DON and 3ANX (r2 = 0.89). This study highlights the first large identification of 3ANX- and NX-producing isolates of F. graminearum in Western Canada. In addition, it is the first identification of 15ADON chemotypes producing 3ANX in Western Canada and the first identification of 3ANX and NX-producing isolates in Manitoba, collected from wheat samples.

Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

Hypoxia is a common feature of glioblastoma (GBM). Circular RNAs (circRNAs) are identified as regulators in cancers. However, the role of circRNAs in GBM remains elusive. Here, circPLOD2a and circPLOD2b, spliced from the same parental gene PLOD2, are identified as hypoxia-responsive circRNAs. Overexpression of circPLOD2a and b enhance while knockdown inhibit GBM cell aggressiveness. The protein partners and downstream molecules were investigated by RNA-pulldown, mass spectrometry and RNA-seq. Mechanistically, HIF1α induces the expression of circPLOD2a and b, which competitively bind to HuR, causing a degradation of XIRP1 in vitro and in vivo. Clinical data demonstrate circPLOD2a and b are highly expressed in GBM negatively correlated with XIRP1, whose lower expression associates with higher glioma grade and worse prognosis. In conclusion, hypoxia-induced circPLOD2a and b are oncogenic regulators of tumour aggressiveness through attenuating the interaction between HuR and XIRP1 in glioblastoma cells and may be potential therapeutic targets for this disease.

Exploring the potential of MRM-IDA-EPI method in mass spectrometry for exposomic analysis: a commentary.

Exposomics is a field that studies environmental exposures and their impact on human health. The MRM-IDA-EPI method, which combines targeted and untargeted mass spectrometry methods, is useful for identifying and quantifying biomarkers in various biological matrices. The method's accuracy and precision in forensic toxicological screening suggest potential applications for detecting low-level environmental exposures. It can help detect and understand environmental exposures, explain their metabolic processes, and assess their impact on human health more effectively.

Lysine succinylome analysis of MRSA reveals critical roles in energy metabolism and virulence.

MRSA's resistance poses a global health challenge. This study investigates lysine succinylation in MRSA using proteomics and bioinformatics approaches to uncover metabolic and virulence mechanisms, with the goal of identifying novel therapeutic targets. Mass spectrometry and bioinformatics analyses mapped the MRSA succinylome, identifying 8 048 succinylation sites on 1 210 proteins. These analyses included Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) network construction (e.g. using the STRING database), providing a comprehensive functional and interactive landscape of succinylated proteins. The succinylated proteins were predominantly involved in cytoplasmic metabolic processes, with enrichment in glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle. Both of these pathways are critical for MRSA's energy production, growth, and virulence, supplying the necessary metabolic intermediates and energy to support bacterial survival and pathogenicity. Motif analysis revealed 13 conserved motifs, while PPI analysis highlighted FnbA as a central virulence factor. Succinylation significantly influences MRSA's metabolism and virulence, potentially impacting biofilm by modifying key proteins such as FnbA, bifunctional autolysin, and LuxS. These findings provide new avenues for developing antibiofilm strategies and therapeutic interventions against MRSA.

Rainy and Dry Seasons Are Relevant Factors Affecting Chemical and Antioxidant Properties of Meliponini Honey

Brazilian stingless bee species produce honey with distinct physicochemical and bioactive properties shaped by environmental factors. This study investigated the effects of the rainy and dry seasons on the physicochemical characteristics, chemical fingerprinting, mineral content, and antioxidant capacity of honey from Melipona mondury and Melipona bicolor. The honey samples were analyzed for their phytochemical properties (official methods), total phenolics (Folin–Ciocalteu method), flavonoid content (aluminum complex formation method), antioxidant capacity (FRAP and ABTS assays), and antioxidant activity (erythrocyte model). The mineral content was assessed via TXRF spectroscopy, and chemical fingerprinting was conducted using mass spectrometry. Chemometric tools were used for the samples’ discriminating analyses, including Principal Component Analysis (PCA) and Partial Least Squares–Discriminant Analysis (PLS-DA). Seasonal variations significantly affected the moisture, total soluble solids, and acidity. In turn, the antioxidant capacity was influenced mainly by the bee species. The mineral composition, particularly potassium, phosphorus, and calcium, remained stable. Multivariate analysis identified m/z ions (VIP scores > 2.5), rather than physicochemical or antioxidant capacity parameters, as critical for seasonal discrimination. The antioxidant activity, assessed by oxidative hemolysis prevention, was robust across the seasons, with M. mondury honey (2 mg·mL−1) from the rainy season outperforming ascorbic acid. These findings underscore the impact of the rainy and dry seasons and the potential of secondary metabolite fingerprinting to identify collection periods.

Gold-Modified Covalent Organic Frameworks-Assisted Laser Desorption/Ionization Mass Spectrometry for Analysis of Metabolites Induced by Triclosan Exposure.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) holds great promise for the rapid and sensitive detection of biomolecules, but its precise detection of small molecule metabolites is hindered by severe background interference from the organic matrix in the low molecular weight range. To address this issue, nanomaterials have commonly been utilized as substrates in LDI-MS. Among them, covalent organic frameworks (COFs), known for their unique optical absorption and structural properties, have garnered significant attention. Despite these advantages, their low ionization efficiency remains a challenge. Herein, a composite material of COF-S@Au nanoparticles (NPs), by incorporating Au NPs into a sulfur-functionalized COF (COF-S) through postsynthetic modification, was designed and adopted as substrates. This hybrid material leverages the synergistic effects of COF-S and Au NPs to improve the desorption/ionization efficiency and minimize background interference. The COF-S@Au NPs demonstrated a 5-16-fold improvement in MS signals of small biomolecules along with a clean background and excellent resistance to salt and protein interference. Their corresponding limits of detection (LODs) were achieved at ∼pmol. Furthermore, the COF-S@Au NPs were applied to analyze metabolites in a triclosan (TCS)-exposed mouse model, successfully identifying 10 differential metabolites associated with TCS toxicity. This work provides a foundation for developing advanced LDI-MS materials for high-performance metabolic analysis and offers valuable insights into TCS metabolic toxicity with potential applications in environmental toxicology.

Antimicrobial resistance profiles and molecular epidemiology of Klebsiella pneumoniae isolates from Scottish bovine mastitis cases.

Klebsiella pneumoniae are opportunistic pathogens which can cause mastitis in dairy cattle. K. pneumoniae mastitis often has a poor cure rate and can lead to the development of chronic infection, which has an impact on both health and production. However, there are few studies which aim to fully characterize K. pneumoniae by whole-genome sequencing from bovine mastitis cases. Here, K. pneumoniae isolates associated with mastitis in dairy cattle were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing. Furthermore, whole-genome sequence data were used for phylogenetic analyses and both virulence and antimicrobial resistance (AMR) prediction, in parallel with phenotypic AMR testing. Forty-two isolates identified as K. pneumoniae were subject to whole-genome sequencing, with 31 multi-locus sequence types being observed, suggesting the source of these isolates was likely environmental. Isolates were examined for key virulence determinants encoding acquired siderophores, colibactin, and hypermucoidy. The majority of these were absent, except for ybST (encoding yersiniabactin) which was present in six isolates. Across the dataset, there were notable levels of phenotypic AMR against streptomycin (26.2%) and tetracycline (19%), and intermediate susceptibility to cephalexin (26.2%) and neomycin (21.4%). Of importance was the detection of two ESBL-producing isolates, which demonstrated multi-drug resistance to amoxicillin-clavulanic acid, streptomycin, tetracycline, cefotaxime, cephalexin, and cefquinome.

An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF).

Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms; however, it is challenging to implement in routine settings. In this protocol, we combine a robust chromatographic platform with a high-performance mass spectrometric setup to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients (Evosep One) combined with a trapped ion mobility time-of-flight mass spectrometer (timsTOF, Bruker). The timsTOF enables parallel accumulation-serial fragmentation (PASEF), in which ions are accumulated and separated by their ion mobility, maximizing ion usage and simplifying spectra. Combined with data-independent acquisition (DIA), it offers high peak sampling rates and near-complete ion coverage. Here, we explain how to balance quantitative accuracy, specificity, proteome coverage and sensitivity by choosing the best PASEF and DIA method parameters. The protocol describes how to set up the liquid chromatography-mass spectrometry system and enables PASEF method generation and evaluation for varied samples by using the py_diAID tool to optimally position isolation windows in the mass-to-charge and ion mobility space. Biological projects (e.g., triplicate proteome analysis in two conditions) can be performed in 3 d with ~3 h of hands-on time and minimal marginal cost. This results in reproducible quantification of 7,000 proteins in a human cancer cell line in quadruplicate 21-min injections and 29,000 phosphosites for phospho-enriched quadruplicates. Synchro-PASEF, a highly efficient, specific and novel scan mode, can be analyzed by Spectronaut or AlphaDIA, resulting in superior quantitative reproducibility because of its high sampling efficiency.

Sensitive and Deep Coverage Phosphoproteome Detection Method Reveals Spleen as a More Sensitive Organ than Liver in Early Detection of Liver Fibrosis-Related Signaling Protein Dysregulation.

Although cathepsin S is transported from the spleen to the liver, where it cleaves collagen XVIII to produce endostatin and plays a critical role in the onset of early liver fibrosis, the relationship between liver fibrosis and spleen function remains underexplored. Given the roles of phosphorylation in disease, understanding its regulatory mechanism in early liver fibrosis is crucial. Despite advances in mass spectrometry enhancing phosphoproteomics, its application is limited by small clinical samples and subtle protein changes. We optimized a phosphoproteomic workflow, adjusting the protein amounts and using different enrichment beads and varied mass spectrometers, achieving deep phosphoproteomic coverage from minimal samples. We identified over 46,000 phosphosites in HepG2 cells and over 29,000 phosphosites in mouse liver samples using just 500 μg of proteins. Even with as little as 50 μg of 293T proteins, we detected over 11,000 phosphosites, 1.2 times more than the recently reported RUPE-phospho. Using the Sensitive and Deep Coverage Phosphoproteome Detection method, abbreviated as SDC-PhosDet, we demonstrated that in early liver fibrosis, the spleen exhibits more rapid and sensitive phosphorylation changes than the liver, affecting proteins closely linked to signaling and metabolism such as STAT1, JUN, CBL, ATP7B, and PTPN2. These findings highlight the spleen's role and offer new avenues for investigating the molecular mechanisms of early liver fibrosis, diagnosis, and intervention beyond the liver itself. Moreover, this method holds promise for applying phosphoproteomics to early-stage liver fibrosis using clinical microsamples.

Proteomic Profiling Reveals Alterations in Metabolic and Cellular Pathways in Severe Obesity and Following Metabolic Bariatric Surgery.

In this study, we investigated the impact of bariatric surgery on the adipose proteome to better understand the metabolic and cellular mechanisms underlying weight loss following the procedure. A total of 46 patients with severe obesity were included, with samples collected both before and after bariatric surgery. Additionally, 15 healthy, non-obese individuals who did not undergo surgery served as controls and were studied once. We utilized quantitative liquid chromatography tandem mass spectrometry analysis to conduct a large-scale proteomic study on abdominal subcutaneous biopsies obtained from the study participants. Our proteomic profiling revealed that among the 2254 compared proteins, 46 were upregulated, and 34 were downregulated 6 months post-surgery compared to baseline (FDR < 0.01). We observed a downregulation of proteins associated with mitochondrial integrity, amino acid catabolism, and lipid metabolism in the patients with severe obesity compared to the controls. Bariatric surgery was associated with an upregulation in pathways related to mitochondrial function, protein synthesis, folding and trafficking, actin cytoskeleton regulation, and DNA binding and repair. These findings emphasize the significant changes in metabolic and cellular pathways following bariatric surgery, highlighting the potential mechanisms underlying the observed health improvements post-bariatric surgery. The data provided alongside this paper will serve as a valuable resource for the development of targeted therapeutic strategies for obesity and related metabolic complications.

Unraveling the Stoichiometric Interactions and Synergism between Ligand-Protected Gold Nanoparticles and Proteins.

Nanomaterials that engage in well-defined and tunable interactions with proteins are pivotal for the development of advanced applications. Achieving a precise molecular-level understanding of nano-bio interactions is essential for establishing these interactions. However, such an understanding remains challenging and elusive. Here, we identified stoichiometric interactions of water-soluble gold nanoparticles (Au NPs) with bovine serum albumin (BSA), unraveling their synergism in manipulating emission of nano-bio conjugates in the second near-infrared (NIR-II) regime. Using Au25(p-MBS)18 (p-MBS = para-mercaptobenzenesulfonic acid) as paradigm particles, we achieved precise binding of Au NPs to BSA with definitive molar ratios of 1:1 and 2:1, which is unambiguously evidenced by high-resolution mass spectrometry and transmission electron microscopy. Molecular dynamics simulations identified well-defined binding sites, mediated by electrostatic interactions and hydrogen bonds between the p-MBS moieties on the Au25(p-MBS)18 surface and BSA. Particularly, positively charged residues on BSA were found to be pivotal. By careful control of the molar ratio of Au25(p-MBS)18 to BSA, atomically precise [Au25(p-MBS)18]x-BSA conjugates (x = 1 or 2) could be formed. Through a comprehensive spectroscopy study, an electron transfer process and synergistic effect were manifested in the Au25(p-MBS)18-BSA conjugates, leading to drastically enhanced emission in the NIR-II window. This work offers insights into the precise engineering of nanomaterial-protein interactions and opens new avenues for the development of next-generation nano-bio conjugates for nanotheranostics.