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The kinetic energy dependence of the title reaction is examined using guided ion beam tandem mass spectrometry. Because this reaction is spin-forbidden, crossings between octet and sextet hypersurfaces presumably must occur. Furthermore, Sm+ must transition from a 4f66s1 configuration in the reactant to 4f55d2 in order to have the orbital occupancy required to form the triple bond in SmO+ (6Δ). Despite being strongly exothermic (∼4 eV), the reaction proceeds with low efficiency (18% ± 4%) via a barrierless process at low energies. Below ∼0.3 eV, the cross section follows a kinetic energy dependence that roughly parallels that of the collision rate for ion–dipole reactions. At higher collision energies, the reaction cross section increases until it follows the trajectory cross section closely from 3 to 5 eV, indicating that another pathway opens on the reaction hypersurface. Modeling this increase yields a threshold energy for this new pathway at 0.54 ± 0.05 eV. Theoretical potential energy surfaces that do not include spin–orbit interactions for the reaction show that there is a barrier of height 1.19 eV (MP2) or 0.49 eV [CCSD(T)] to insertion of Sm+ into the N2–O bond and that there are several places where octet and sextet surfaces can intersect and interact. By considering the distribution of spin–orbit states generated in the ion source, the internal energy of the N2O reactant, and the influence of coupling between electronic, orbital, and rotational angular momentum, the low-efficiency, exothermic behavior as well as the increase in efficiency at higher energies can plausibly be explained. [ABSTRACT FROM AUTHOR]

The kinetic energy dependence of the title reaction is examined using guided ion beam tandem mass spectrometry. Because this reaction is spin-forbidden, crossings between octet and sextet hypersurfaces presumably must occur. Furthermore, Sm+ must transition from a 4f66s1 configuration in the reactant to 4f55d2 in order to have the orbital occupancy required to form the triple bond in SmO+ (6Δ). Despite being strongly exothermic (∼4 eV), the reaction proceeds with low efficiency (18% ± 4%) via a barrierless process at low energies. Below ∼0.3 eV, the cross section follows a kinetic energy dependence that roughly parallels that of the collision rate for ion–dipole reactions. At higher collision energies, the reaction cross section increases until it follows the trajectory cross section closely from 3 to 5 eV, indicating that another pathway opens on the reaction hypersurface. Modeling this increase yields a threshold energy for this new pathway at 0.54 ± 0.05 eV. Theoretical potential energy surfaces that do not include spin–orbit interactions for the reaction show that there is a barrier of height 1.19 eV (MP2) or 0.49 eV [CCSD(T)] to insertion of Sm+ into the N2–O bond and that there are several places where octet and sextet surfaces can intersect and interact. By considering the distribution of spin–orbit states generated in the ion source, the internal energy of the N2O reactant, and the influence of coupling between electronic, orbital, and rotational angular momentum, the low-efficiency, exothermic behavior as well as the increase in efficiency at higher energies can plausibly be explained. [ABSTRACT FROM AUTHOR]

Glucagon plays a central role in amino acid (AA) homeostasis. The dog is an established model of glucagon biology, and recently, metabolomic changes in people associated with glucagon infusions have been reported. Glucagon also has effects on the kidney; however, changes in urinary AA concentrations associated with glucagon remain under investigation. Therefore, we aimed to fill these gaps in the canine model by determining the effects of glucagon on the canine plasma metabolome and measuring urine AA concentrations. Employing two constant rate glucagon infusions (CRI) - low-dose (CRI-LO: 3 ng/kg/min) and high-dose (CRI-HI: 50 ng/kg/min) on five research beagles, we monitored interstitial glucose and conducted untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) on plasma samples and urine AA concentrations collected pre- and post-infusion. The CRI-HI induced a transient glucose peak (90-120 min), returning near baseline by infusion end, while only the CRI-LO resulted in 372 significantly altered plasma metabolites, primarily reductions (333). Similarly, CRI-HI affected 414 metabolites, with 369 reductions, evidenced by distinct clustering post-infusion via data reduction (PCA and sPLS-DA). CRI-HI notably decreased circulating AA levels, impacting various AA-related and energy-generating metabolic pathways. Urine analysis revealed increased 3-methyl-l-histidine and glutamine, and decreased alanine concentrations post-infusion. These findings demonstrate glucagons glucose-independent modulation of the canine plasma metabolome and highlight the dogs relevance as a translational model for glucagon biology. Understanding these effects contributes to managing dysregulated glucagon conditions and informs treatments impacting glucagon homeostasis.

Monofluoroacetate is a highly lethal toxin that causes death by inhibiting cellular adenosine triphosphate (ATP) production. The heart and brain are the primary target organs. Acute death is attributed to cardiac fibrillation and/or convulsions. Although it occurs naturally in some plants, a major source of animal intoxication is access to sodium monofluoroacetate (NaMFA) pesticide, which continues to be a concern in the USA and around the world despite restricted use in some countries including the USA. There are also concerns about misuse of this pesticide for malicious poisoning. Currently, a tissue-based diagnostic method for NaMFA intoxication in animals is lacking. There is a critical need by the veterinary diagnostic community for a simple, sensitive and reliable tissue-based diagnostic test to confirm NaMFA poisoning in animals. We have developed and extensively evaluated a sensitive novel liquid chromatography combined with tandem mass spectrometry method suitable for this purpose. The limits of detection and limits of quantitation are 1.7 and 5.0 ng/g, respectively. The accuracy and precision met or exceeded expectations. The method performance was verified using the incurred kidney obtained from animal diagnostic cases. This novel kidney-based method is now available for clinical use and can help with diagnostic purposes, including detecting potential issues related to animal foods.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

Isomerized amino acid residues have been identified in many peptides extracted from tissues or excretions of humans and animals. These isomerized residues can play key roles by affecting biological activity or by exerting an influence on the process of aging. Isomerization occurs spontaneously and does not result in a mass shift. Thus, identifying and localizing isomerized residues in biological samples is challenging. Herein, we introduce a fast and efficient method using tandem mass spectrometry (MS) to locate isomerized residues in peptides. Although MS2 spectra are useful for identifying peptides that contain an isomerized residue, they cannot reliably localize isomerization sites. We show that this limitation can be overcome by utilizing MS3 experiments to further evaluate each fragment ion from the MS2 stage. Comparison at the MS3 level, utilizing statistical analyses, reveals which MS2 fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS2 product ions by collision cross-section. The MS3 approach can be implemented using either ion-trap or beam-type collisional activation and is compatible with the quantification of isomer mixtures when coupled to a calibration curve. The method can also be implemented in combination with liquid chromatography in a targeted approach. Enabling the identification and localization of isomerized residues in peptides with an MS-only methodology will expand accessibility to this important information.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

This study investigates the effects of kinetic energy and projectile size on particle emission from a phenylalanine overlayer adsorbed on a graphene bilayer during argon projectile bombardment. Projectiles of various sizes (1–1000 atoms) and kinetic energies (0.5–40 keV) are used to study their impact on the ejection efficiency, mass distribution, and kinetic and angular spectra of the emitted substances. Particle ejection mechanisms are discussed in scenarios with and without sample perforation. Additionally, this study investigated the potential use of graphene in secondary ion mass spectrometry or neutral secondary mass spectrometry analyses. [ABSTRACT FROM AUTHOR]

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

Here, we present the second generation of our bicyclic peptide library (NTB), featuring a stereodiversified structure and a simplified construction strategy. We utilized a tandem ring-opening metathesis and ring-closing metathesis reaction (ROM-RCM) to cyclize the linear peptide library in a single step, representing the first reported instance of this reaction being applied to the preparation of macrocyclic peptides. Moreover, the resulting bicyclic peptide can be easily linearized for MS/MS sequencing with a one-step deallylation process. We employed this library to screen against the E363-R378 epitope of MYC and identified several MYC-targeting bicyclic peptides. Subsequent in vitro cell studies demonstrated that one candidate, NT-B2R, effectively suppressed MYC transcription activities and cell proliferation.

BACKGROUND: A better understanding of treatment progression and recovery in pulmonary tuberculosis (TB) infectious disease is crucial. This study analyzed longitudinal serum samples from pulmonary TB patients undergoing interventional treatment to identify surrogate markers for TB-related outcomes. METHODS: Serum that was collected at baseline and 8, 17, 26, and 52 weeks from 30 TB patients experiencing durable cure were evaluated and compared using a sensitive LC-MS/MS proteomic platform for the detection and quantification of differential host protein signatures relative to timepoint. The global proteome signature was analyzed for statistical differences across the time course and between disease severity and treatment groups. RESULTS: A total of 676 proteins showed differential expression in the serum over these timepoints relative to baseline. Comparisons to understand serum protein dynamics at 8 weeks, treatment endpoints at 17 and 26 weeks, and post-treatment at 52 weeks were performed. The largest protein abundance changes were observed at 8 weeks as the initial effects of antibiotic treatment strongly impacted inflammatory and immune modulated responses. However, the largest number of proteome changes was observed at the end of treatment time points 17 and 26 weeks respectively. Post-treatment 52-week results showed an abatement of differential proteome signatures from end of treatment, though interestingly those proteins uniquely significant at post-treatment were almost exclusively downregulated. Patients were additionally stratified based upon disease severity and compared across all timepoints, identifying 461 discriminating proteome signatures. These proteome signatures collapsed into discrete expression profiles with distinct pathways across immune activation and signaling, hemostasis, and metabolism annotations. Insulin-like growth factor (IGF) and Integrin signaling maintained a severity signature through 52 weeks, implying an intrinsic disease severity signature well into the post-treatment timeframe. CONCLUSION: Previous proteome studies have primarily focused on the 8-week timepoint in relation to culture conversion status. While this study confirms previous observations, it also highlights some differences. The inclusion of additional end of treatment and post-treatment time points offers a more comprehensive assessment of treatment progression within the serum proteome. Examining the expression dynamics at these later time periods will help in the investigation of relapse patients and has provided indicative markers of response and recovery.

Antisense oligonucleotides (ASOs) are crucial for biological applications as they bind to complementary RNA sequences, modulating protein expression. ASOs undergo synthetic modifications like phosphorothioate (PS) backbone and locked nucleic acid (LNA) to enhance stability and specificity. Tandem mass spectrometry (MS) techniques were employed to study gapmer ASOs, which feature a DNA chain within RNA segments at both termini, revealing enhanced cleavages with ultraviolet photodissociation (UVPD) and complementary fragment ions from collision-induced dissociation (CID) and electron detachment dissociation (EDD). 2DMS, a data-independent analysis technique, allowed for comprehensive coverage and identification of shared fragments across multiple precursor ions. EDD fragmentation efficiency correlated with precursor ion charge states, with higher charges facilitating dissociation due to intramolecular repulsions. An electron energy of 22.8 eV enabled electron capture and radical-based cleavage. Accumulating multiple scans and generating average spectra improved signal intensity, aided by denoising algorithms. Data analysis utilised a custom Python script capable of handling modifications and generating unique mass lists. [ABSTRACT FROM AUTHOR]

ABSTRACT The formulation of multi‐nutrient blocks based on low‐cost and locally available browse feed resources can be a valid feeding strategy in Sub‐Saharan Africa, where inadequate feed supply, both in quality and quantity, is a major constraint. We evaluated the four different inclusion percentages (M‐0%, M‐25%, M‐35% and M‐45%) of Moringa stenopetala leaf powder to multi‐nutrient blocks on their change on blood metabolite of dairy cows under practical, ranging conditions. Multi‐nutrient blocks with four inclusion rates of M. stenopetala leaves were applied as complementary feed for free ranging dairy cows. The study was performed on 24 free ranging dairy cows reared around Arba Minch town in the Southern Ethiopian Rift Valley. Blood samples were collected from the jugular vein of dairy cows both before and after supplementation. Plasma glucose, beta hydroxy butyrate (BHB), urea, creatinine, triglycerides and nonesterified fatty acids (NEFA) concentration was quantified spectrophotometrically. Dried serum spots were subject to quantitative electrospray tandem mass spectrometry to estimate changes in nutrient metabolism based on selected carnitines. Based on these measurements, the milk yield and body condition score were increased during the period of multi‐nutrient block supplementation. During the supplementation period, the cows got higher plasma glucose, triglyceride and urea concentrations and lower concentrations of BHB, NEFA and creatinine. From the metabolite profiles, a more efficient nutrient use could be concluded. Although no clear dose−response relationship was observed, the highest inclusion of the M. stenopetala leaves in the multi‐nutrient blocks gave the best performance. This outcome supports the idea of implementing M. stenopetala based multi‐nutrient blocks on tropical smallholder farms that are not easily accessible to conventional extension services. [ABSTRACT FROM AUTHOR]

Rationale: Optimizing the structure of digital polymers is an efficient strategy to ensure their tandem mass spectrometry (MS/MS) readability. In block‐truncated poly(phosphodiester)s, homolysis of C–ON bonds in long chains permits the release of smaller blocks amenable to sequencing. Yet the dissociation behavior of diradical blocks was observed to strongly depend on their charge state. Methods: Polymers were ionized in negative mode electrospray and activated in‐source so that blocks released as primary fragments can be investigated using ion mobility spectrometry (IMS) or sequenced in the post‐IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions using the IMSCal software. A multistep protocol based on quantum methods and classical molecular dynamics was implemented for molecular modeling and calculation of theoretical CCS. Results: Unlike their triply charged homologues, dissociation of diradical blocks at the 2– charge state produces additional fragments, with +1 m/z shift for those holding the nitroxide α‐termination and −1 m/z for those containing the carbon‐centered radical ω‐end. These results suggest cyclization of these diradical species, followed by H• transfer on activated reopening of this cycle. This assumption was validated using IMS resolution of the cyclic/linear isomers and supported by molecular modeling. Conclusions: Combining IMS with molecular modeling provided new insights into how the charge state of digital blocks influences their dissociation. These results permit to define new guidelines to improve either ionization conditions or the structural design of these digital polymers for best MS/MS readability. [ABSTRACT FROM AUTHOR]

Rationale: Sphingomyelins (SMs) and resulting metabolic products serve important functional and cell signaling roles and can act as potential biomarkers and therapeutic targets in many pathological disorders. SMs each contain a sphingoid base, an amide‐linked fatty acyl chain, and a phosphocholine headgroup. Despite these simple building blocks, variations and modifications of both the sphingoid base and the fatty acyl chain result in a diverse array of structurally complicated SM compounds. Conventional tandem mass spectrometry (MS/MS) using the collision‐induced dissociation (CID) method only provides limited structural information, necessitating other tools to unravel the structural complexity of these lipids. Methods: We utilize electron‐induced dissociation (EID) and sequential CID/EID approaches to elucidate detailed structural features of SMs. Integrating the CID/EID method into an imaging MS workflow enables accurate identification of SMs directly from kidney tissue. Results: The application of EID enables identification of SMs at the molecular species level, identifying the sphingosine base and the amide‐linked fatty acyl chains. Furthermore, removal of the phosphocholine headgroup via CID followed by sequential EID in an MS3 analysis (CID/EID) enhances the structural information obtained. CID/EID provides diagnostic fragmentation patterns revealing the hydroxylation site and double bond position in both the sphingosine base and amide‐linked fatty acyl chains. Conclusions: Detailed structural information of SMs from synthetic standards and biological tissue samples is obtained using an alternative electron‐based dissociation method. Accurate characterization of SMs promises to better inform studies of tissue biochemistry, lipid metabolism, and molecular pathology. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) in the air are one of the major concerns due to their possible health risks. In addition, airborne microplastics act as a carrier for several toxic air pollutants and cause carcinogenic, mutagenic and teratogenic effects. Nylon 66 is one of the most abundant MPs in the microenvironments. In this study, we report an optimized method for the quantification of nylon 66 MPs present in the fine particulate matter (PM2.5) using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The optimized method exhibited excellent linearity, recovery rates, intraday and interday precision and sensitivity. The optimized method was deployed to carry out a pilot study to measure the mass concentrations of nylon 66 microplastics present in the inhalable fraction of particulate matter collected in a shopping complex. The observed mass concentrations of nylon 66 MPs in this study are in the range from 0.30 ng m−3 to 4.37 ng m−3. This study demonstrates the presence of inhalable fraction of nylon 66 MPs in the indoor environment and their mass concentrations were determined using HPLC-MS/MS. [ABSTRACT FROM AUTHOR]

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2–5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants. [ABSTRACT FROM AUTHOR]

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease worldwide. While injury to the podocytes, visceral epithelial cells that comprise the glomerular filtration barrier, drives albuminuria, proximal tubule (PT) dysfunction is the critical mediator of DKD progression. Here, we report that the podocyte-specific induction of human KLF6, a zinc-finger binding transcription factor, attenuates podocyte loss, PT dysfunction, and eventual interstitial fibrosis in a male murine model of DKD. Utilizing combination of snRNA-seq, snATAC-seq, and tandem mass spectrometry, we demonstrate that podocyte-specific KLF6 triggers the release of secretory ApoJ to activate calcium/calmodulin dependent protein kinase 1D (CaMK1D) signaling in neighboring PT cells. CaMK1D is enriched in the first segment of the PT, proximal to the podocytes, and is critical to attenuating mitochondrial fission and restoring mitochondrial function under diabetic conditions. Targeting podocyte-PT signaling by enhancing ApoJ-CaMK1D might be a key therapeutic strategy in attenuating the progression of DKD. While diabetic kidney disease is characterized by initial glomerular injury, proximal tubule dysfunction determines the progression to kidney fibrosis. Here, the authors show that podocyte KLF6 attenuates proximal tubule injury via ApoJ-CaMK1D signaling in a murine model of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

AbstractA method for the determination of eight benzenes (BTEXs) and twelve chlorobenzenes (CBs) in goat’s milk by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS/MS) was developed. The study investigated the impact of various factors such as extraction fiber type, salt amount, equilibrium conditions, and desorption conditions on the outcomes. Target analytes were separated on a DB-HeavyWAX column and quantified using the external standard method. The results showed that the target compounds had a good linear relationship in the range of 0.01 ∼ 50 μg/L (R2 > 0.997), the limit of detection (LOD) was 0.003 ∼ 0.150 μg/L, and the limit of quantification (LOQ) was 0.01 ∼ 0.50 μg/L. The average recoveries were 82%–116% and the relative standard deviation (RSD) was 0.8%–17.3% under the three addition levels of 1×, 2×, and 10 × LOQ. In a survey of twenty goat’s milk samples, only ethylbenzene, xylenes, cumene, chlorobenzene, and 1,4-dichlorobenzene were detected at levels exceeding their respective limits of quantification. The method was evaluated using two ecological scales (Eco-Scale), GAPI and AGREEN, to verify its environmental friendliness and applicability. This method is simple, green, and efficient, which provides a certain theoretical basis for the production and quality safety evaluation of dairy products. [ABSTRACT FROM AUTHOR]

Background: Podocyte injury is a common pathologic mechanism in diabetic kidney disease (DKD) and obesity-related glomerulopathy (ORG). Our previous study confirmed that Inonotus obliquus (IO) improved podocyte injury on DKD rats. The current study explored the pharmacological effects, related mechanisms and possible active components of IO on ORG mice. Methods: Firstly, by combining ultra-high performance liquid chromatography tandem mass spectrometry analysis (UPLC-Q-TOF-MS) with network pharmacology to construct the human protein-protein interaction mechanism and enrich the pathway, which led to discover the crucial mechanism of IO against ORG. Then, ORG mice were established by high-fat diet and biochemical assays, histopathology, and Western blot were used to explore the effects of IO on obesity and podocyte injury. Finally, network pharmacology-based findings were confirmed by immunohistochemistry. The compositions of IO absorbed in mice plasma were analyzed by UPLC-Q-TOF-MS and molecular docking was used to predict the possible active compounds. Results: The network pharmacology result suggested that IO alleviated the inflammatory response of ORG by modulating TNF signal. The 20-week in vivo experiment confirmed that IO improved glomerular hypertrophy, podocyte injury under electron microscopy, renal nephrin, synaptopodin, TNF-α and IL-6 expressions with Western blotting and immunohistochemical staining. Other indicators of ORG such as body weight, kidney weight, serum total cholesterol, liver triglyceride also improved by IO intervention. The components analysis showed that triterpenoids, including inoterpene F and trametenolic acid, might be the pharmacodynamic basis. Conclusion: The research based on UPLC-Q-TOF-MS analysis, network pharmacology and in vivo experiment suggested that the amelioration of IO on podocyte injury in ORG mice via its modulation on TNF signal. Triterpenoids were predicated as acting components. [ABSTRACT FROM AUTHOR]

Background: Head and neck paragangliomas (HNPGLs) are rare neuroendocrine tumors that pose significant challenges in both diagnosis and treatment. The pathogenic mechanism remains unclear, and there is no proteomic analysis-based molecular classification. Therefore, gaining a deeper understanding of this disease from the protein level is crucial because proteins play a fundamental role in the occurrence and development of tumors. Methods: We collected 44 tumor samples from patients diagnosed with HNPGL. The adrenal paraganglioma tissue (N = 46) was used as the disease control group and the chorda tympani nerves (N = 18) were used as the control group. High-pH reversed-phase liquid chromatography and liquid chromatography with tandem mass spectrometry analyses were used to build an integrated protein database of tumor samples. We then obtained two sets of differentially expressed proteins between the tumor group and the control group to identify the unique proteomic signatures of HNPGLs. Ingenuity pathway analysis annotations were used to perform the functional analysis. Subsequently, we developed a clinically relevant molecular classification for HNPGLs that connected the clinical characteristics with meaningful proteins and pathways to explain the varied clinical manifestations. Results: We identified 6,640 proteins in the HNPGL group, and 314 differentially expressed proteins unique to HNPGL were discovered via inter-group comparison. We identified two HNPGL subgroups that significantly differed in clinical manifestation and proteomic characteristics. On the basis of the proteomic results, we proposed a pathogenic mechanism underlying HNPGL. Conclusion: We conducted a comprehensive analysis of the molecular mechanisms of HNPGL to build, for the first time, a clinically relevant molecular classification. By focusing on differential proteomic analyses between different types of paragangliomas, we were able to obtain a comprehensive description of the proteomic characteristics of HNPGL, which will be valuable for the search for significant biomarkers as a new treatment method for HNPGL. [ABSTRACT FROM AUTHOR]

Introduction: Epididymal lumen fluids provides a stable microenvironment for sperm maturation. Ca2+ binding protein CABS1 is known to maintain structural integrity of mouse sperm flagella during epididymal transit of sperm. Besides, CABS1 was reported to contain anti-inflammatory peptide sequences and be present in both human saliva and plasma. However, little is known about the role of CABS1 in regulation of the microenvironment of epididymal lumen fluids.Methods: To further confirm the role of CABS1 in epididymis, we identified the expression of CABS1 in epididymal lumen fluids. Moreover, high performance liquid chromatography, coupled with tandem mass spectrometry technique was used to analyze the metabolic profiles and in vivo microperfusion of the cauda epididymis and inductively coupled plasma mass spectrometry (ICP-MS) assays was used to detect the concentration of metal ion of mouse cauda epididymal lumen fluids in CABS1 deficient and normal mice.Results: The results showed that CABS1 is present in epididymal lumen fluids, and the concentration of calcium in epididymal lumen fluids is not changed in Cabs1-/- male mice. Among 34 differential metabolites identified in cauda epididymis, 21 were significantly upregulated while 13 were significantly downregulated in KO cauda epididymis. Pathway analysis identified pyrimidine metabolism, inositol phosphate metabolism, arachidonic acid metabolism, purine metabolism and histidine metabolism as relevant pathways in cauda epididymis.Discussion: The perturbations of mitochondrial dysfunction and inflammation may be the crucial reason for the poor performance of Cabs1-/- sperm. [ABSTRACT FROM AUTHOR]

Background: Bovine genital campylobacteriosis (BGC) is a globally important venereal disease of cattle caused by Campylobacter fetus subspecies venerealis. Diagnosis of BGC is highly challenging due to the lack of accurate diagnostic tests. Methods: To characterise the biomarkers for C. fetus venerealis infection, a total of twelve cycling heifers were selected and categorised as vaccinated (n = 6) with Vibrovax® (Zoetis™) and unvaccinated (n = 6). All heifers were oestrous synchronised with a double dose of prostaglandin (PGF2α) 11 days apart and when in oestrous intravaginally challenged with 2.7 x 109 CFU live C. fetus venerealis. DNA extracted from vaginal mucus samples was screened using a C. fetus qPCR and 16S rRNA was characterised using Illumina sequencing (V5-V8 region). Relative abundances of serum proteins were calculated using sequential window acquisition of all theoretical fragment ion spectra coupled to tandem mass spectrometry (SWATH-MS) for all heifers at three timepoints: pre-challenge, post-challenge and post-recovery. Results: In 16S rRNA sequencing of vaginal mucus, Campylobacter spp. appeared two days following challenge in unvaccinated compared to 14 days in vaccinated animals, consistent with the qPCR results. Increased relative abundances of Firmicutes and Campylobacterota were identified after C. fetus venerealis challenge and were associated with C. fetus venerealis in vaccinated and unvaccinated heifers. Greater relative abundance of Streptococcus spp. was observed during oestrous rather than dioestrous. In both vaccinated and unvaccinated heifers, Acinetobacter spp. increased after challenge with higher abundance of Corynebacterium spp. in the vaccinated group. A total of 130 unique proteins were identified in SWATH analysis of the serum samples, and the number of differentially abundant proteins found was higher in the vaccinated group after recovery from infection compared to pre-and post-challenge (adjusted P < 0.05 and Log2FC > 0.2). Conclusion: Coglutinin, clusterin, HP homologs, vitamin D binding protein and fetuin B were identified as potential biomarkers for C. fetus venerealis infection and need further study to validate their efficiency as immune biomarkers for BGC. [ABSTRACT FROM AUTHOR]

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid and amino acid oxidation with an incidence of 1 in 200,000 live births. MADD has three clinical phenotypes: severe neonatal-onset with or without congenital anomalies, and a milder late-onset form. Clinical diagnosis is supported by urinary organic acid and blood acylcarnitine analysis using tandem mass spectrometry in newborn screening programs. MADD is an autosomal recessive trait caused by biallelic mutations in the ETFA, ETFB, and ETFDH genes encoding the alpha and beta subunits of the electron transfer flavoprotein (ETF) and ETF-coenzyme Q oxidoreductase enzymes. Despite significant advancements in sequencing techniques, many patients remain undiagnosed, impacting their access to clinical care and genetic counseling. In this report, we achieved a definitive molecular diagnosis in a newborn by combining whole-genome sequencing (WGS) with RNA sequencing (RNA-seq). Whole-exome sequencing and next-generation gene panels fail to detect variants, possibly affecting splicing, in deep intronic regions. Here, we report a unique deep intronic mutation in intron 1 of the ETFDH gene, c.35-959A>G, in a patient with early-onset lethal MADD, resulting in pseudo-exon inclusion. The identified variant is the third mutation reported in this region, highlighting ETFDH intron 1 vulnerability. It cannot be excluded that these intronic sequence features may be more common in other genes than is currently believed. This study highlights the importance of incorporating RNA analysis into genome-wide testing to reveal the functional consequences of intronic mutations. [ABSTRACT FROM AUTHOR]

Background/Objectives: The objective of this study was to investigate the metabolomic profiles of patients with colorectal cancer (CRC) across various stages of the disease. Methods: The plasma samples were obtained from 255 subjects, including patients with CRC in stages I-IV, polyps, and controls. We employed capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography triple quadrupole mass spectrometry to analyze hydrophilic metabolites comprehensively. The data were randomly divided into two groups, and consistent differences observed in both groups were analyzed. Results: Acetylated polyamines, such as N1-acetylspermine and N1, N12-diacetylspermine, consistently showed elevated concentrations in stage IV compared to stages I-III. Non-acetylated polyamines, including spermine and spermidine, exhibited increasing trends from polyp to stage IV. Other metabolites, such as histidine and o-acetylcarnitine, showed decreasing trends across stages. While acetylated polyamines have been reported as CRC detection markers, our findings suggest that they also possess diagnostic potential for distinguishing stage IV from other stages. Conclusions: This study showed stage-specific changes in metabolic profiles, including polyamines, of colorectal cancer. [ABSTRACT FROM AUTHOR]

Early diagnosis of inborn errors of metabolism (IEM) is crucial to ensure early detection of conditions which are treatable. This study reports on targeted metabolomic procedures for the diagnosis of IEM of amino acids, acylcarnitines, creatine/guanidinoacetate, purines/pyrimidines and oligosaccharides, and describes its validation through external quality assessment schemes (EQA). Analysis was performed on a Waters ACQUITY UPLC H-class system coupled to a Waters Xevo triple-quadrupole (TQD) mass spectrometer, operating in both positive and negative electrospray ionization mode. Chromatographic separation was performed on a CORTECS C18 column (2.1 × 150, 1.6 µm). Data were collected by multiple reaction monitoring. The internal and EQA results were generally adequate, with a few exceptions. We calculated the relative measurement error (RME) and only a few metabolites displayed a RME higher than 30 % (asparagine and some acylcarnitine species). For oligosaccharides, semi-quantitative analysis of an educational panel clearly identified the 8 different diseases included. Overall, we have validated our analytical system through an external quality control assessment. This validation will contribute to harmonization between laboratories, thus improving identification and management of patients with IEM. [ABSTRACT FROM AUTHOR]

Anthocyanins (ANCs), a kind of natural pigments, are widely present in food substrates. Evidence has shown that ANCs can promote health in terms of anti-oxidation, anti-tumor, and anti-inflammation. However, the oxidative stability of ANCs limits accurate quantitation and analysis. Therefore, faster, more accurate, and highly sensitive extraction and determination methods are necessary for understanding the role of ANCs in medicine and food. This review presents an updated overview of pretreatment and detection techniques for ANCs in various food substrates since 2015. Liquid-liquid extraction and various green solvent extraction methods, such as accelerated solvents extraction, deep eutectic solvents extraction, ionic liquids extraction, and supercritical fluid extraction, are commonly used pretreatment methods for extraction and purification of ANCs. Liquid chromatography coupled with different detectors (tandem mass spectrometry and UV detectors) and spectrophotometry methods are some of the determination methods for ANC. This study has updated, compared, and discussed different pretreatment and analysis methods. Moreover, the advanced methods and development prospects in this field are comprehensively summarized, which can provide references for further utilization of ANCs. [ABSTRACT FROM AUTHOR]

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a nongenotoxic rat carcinogen, which induced mammary tumors in male and female rats in a 2-yr bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 wk. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary, and liver samples using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as nongenotoxic carcinogen. Mammary DNA samples (n  = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations, namely regions of Apc , Braf , Egfr , Hras , Kras , Nfe2l2 , Pik3ca , Setbp1 , Stk11 , and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error-corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, P  <   0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 wk as compared with 12 wk. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a nongenotoxic carcinogen using a treatment duration as short as 3 months. [ABSTRACT FROM AUTHOR]

We established an efficient analytical approach for simultaneous determination of benthiavalicarb-isopropyl and its four metabolites (KIF-230-M1, KIF-230-M3, KIF-230-M4, and KIF-230-M5) in fruits and vegetables. Samples were extracted with acidified acetonitrile, with octadecylsilane and graphitised carbon black as sorbent for clean-up, and UHPLC-MS/MS for determination. The approach was determined at spikes of 5, 50, and 500 μg kg−1. The recoveries of the six matrices analyzed ranged from 74.5% to 115.6% with 0.5–12.6% for intra-day RSDr (n = 5), and 0.4–13.8% for inter-day RSDR (n = 15). The five analytes in all matrices achieved high linearity (R2 > 0.9917). The limit of quantification (LOQ) of benthiavalicarb-isopropyl as well as its four metabolites in all matrices was 5 μg kg−1. This analytical approach allows for monitoring residues of benthiavalicarb-isopropyl and its metabolites in vegetables and fruits. [ABSTRACT FROM AUTHOR]

This article provides a personal account of the author's career in mass spectrometry, both in the United States and Spain. The author discusses their training in the US and their work with Dr. John Oró at the University of Houston, as well as their experiences with mass spectrometry and gas chromatography. They also mention their involvement in NASA research programs and the social and historic events they witnessed during their time in the US. The article then focuses on the challenges faced by the author in establishing a biomedical mass spectrometry laboratory in Spain, including the lack of support and funding for research. Despite these difficulties, the author and their team managed to publish papers and graduate students. The text also discusses the author's efforts to promote the use of biomedical mass spectrometry in Spain, including organizing international symposiums and meetings. The author reflects on the advancements in mass spectrometry technology during this time period. They also discuss their involvement in the organization of international conferences and the development of biomedical mass spectrometry in Spain. The author served on scientific committees and played a key role in organizing the 15th International Mass Spectrometry Conference in Barcelona in 2000. They also discuss the challenges and successes of their research group, including the creation of a proteomics MS lab. The author reflects on their career in the field of mass spectrometry, particularly in the medical community in Spain. They discuss the challenges they faced in conducting research and the lack of available resources and facilities. [Extracted from the article]

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast‐binding protein, which consists of an approximately 35‐kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). An anti‐GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well‐known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a leading cause of disability and death, and aging is the main nonmodifiable risk factor. Following ischemia, neuroactive steroids have been shown to play a key role in cerebroprotection. Thus, brain steroid concentrations at the time of injury as well as their regulation after stroke are key factors to consider. Here, we investigated the effects of age and cerebral ischemia on steroid levels, behavioral outcomes, and neuronal degeneration in 3- and 18-month-old C57BL/6JRj male mice. Ischemia was induced by middle cerebral artery occlusion for 1 hour followed by reperfusion (MCAO/R) and analyses were performed at 6 hours after MCAO. Extended steroid profiles established by gas chromatography coupled with tandem mass spectrometry revealed that (1) brain and plasma concentrations of the main 5α-reduced metabolites of progesterone, 11-deoxycorticosterone, and corticosterone were lower in old than in young mice; (2) after MCAO/R, brain concentrations of progesterone, 5α-dihydroprogesterone, and corticosterone increased in young mice; and (3) after MCAO/R, brain concentrations of 5α-reduced metabolites of progesterone, 3α5α-tetrahydrodeoxycorticosterone, and 3β5α-tetrahydrodeoxycorticosterone were lower in old than in young mice. After ischemia, old mice showed increased sensori-motor deficits and more degenerating neurons in the striatum than young mice. Altogether, these findings strongly suggest that the decreased capacity of old mice to metabolize steroids toward the 5α-reduction pathway comparatively to young mice may contribute to the worsening of their stroke outcomes. [ABSTRACT FROM AUTHOR]

In this work, more than 200-year-old historical remains of liquorice root powder and a current liquorice root as a reference material were analyzed by HPLC–MS/MS to confirm the botanical authenticity of the historical material by comparing the results obtained. Combining extraction with different solvents with HPLC–MS/MS successfully identified nine chemotaxonomic markers in the historical material, thus confirming its identity as liquorice. In the current reference material, 32 chemotaxonomic markers of liquorice were successfully identified. Therefore, several liquorice-specific compounds in the historical remains analyzed were shown to have degraded over time. In addition, possible fragmentation mechanisms for five liquorice-specific compounds that have not been previously published in the literature were proposed. [ABSTRACT FROM AUTHOR]

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Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Macroautophagy/autophagy is essential for the degradation and recycling of cytoplasmic materials. The initiation of this process is determined by phosphatidylinositol-3-kinase (PtdIns3K) complex, which is regulated by factor BECN1 (beclin 1). UFMylation is a novel ubiquitin-like modification that has been demonstrated to modulate several cellular activities. However, the role of UFMylation in regulating autophagy has not been fully elucidated. Here, we found that VCP/p97 is UFMylated on K109 by the E3 UFL1 (UFM1 specific ligase 1) and this modification promotes BECN1 stabilization and assembly of the PtdIns3K complex, suggesting a role for VCP/p97 UFMylation in autophagy initiation. Mechanistically, VCP/p97 UFMylation stabilizes BECN1 through ATXN3 (ataxin 3)-mediated deubiquitination. As a key component of the PtdIns3K complex, stabilized BECN1 facilitates assembly of this complex. Re-expression of VCP/p97, but not the UFMylation-defective mutant, rescued the VCP/p97 depletion-induced increase in MAP1LC3B/LC3B protein expression. We also showed that several pathogenic VCP/p97 mutations identified in a variety of neurological disorders and cancers were associated with reduced UFMylation, thus implicating VCP/p97 UFMylation as a potential therapeutic target for these diseases. Abbreviation: ATG14:autophagy related 14; Baf A1:bafilomycin A1;CMT2Y: Charcot-Marie-Toothdisease, axonal, 2Y; CYB5R3: cytochromeb5 reductase 3; DDRGK1: DDRGK domain containing 1; DMEM:Dulbecco'smodified Eagle's medium;ER:endoplasmic reticulum; FBS:fetalbovine serum;FTDALS6:frontotemporaldementia and/or amyotrophic lateral sclerosis 6; IBMPFD1:inclusion bodymyopathy with early-onset Paget disease with or withoutfrontotemporal dementia 1; LC-MS/MS:liquid chromatography tandem mass spectrometry; MAP1LC3B/LC3B:microtubule associated protein 1 light chain 3 beta; MS: massspectrometry; NPLOC4: NPL4 homolog, ubiquitin recognition factor;PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3;PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K:phosphatidylinositol 3-kinase; RPL26: ribosomal protein L26; RPN1:ribophorin I; SQSTM1/p62: sequestosome 1; UBA5: ubiquitin likemodifier activating enzyme 5; UFC1: ubiquitin-fold modifierconjugating enzyme 1; UFD1: ubiquitin recognition factor in ERassociated degradation 1; UFL1: UFM1 specific ligase 1; UFM1:ubiquitin fold modifier 1; UFSP2: UFM1 specific peptidase 2; UVRAG:UV radiation resistance associated; VCP/p97: valosin containingprotein; WT: wild-type [ABSTRACT FROM AUTHOR]

Proteome analyses of two contrasting mutant lines and their parent revealed responsive proteins in salt stress. The tandem mass spectrometry analysis resulted in identification of 67 proteins involved in various molecular processes including defense to oxidative stresses. The results demonstrated that total upregulated proteins were similar in sensitive and tolerant mutants (20), but downregulated proteins were significantly higher in sensitive line. Similarly, downregulated proteins were equal in tolerant mutant and wild type (23), though upregulated proteins were much higher than their parent. Although, tolerant, sensitive and their parent had partially the same pattern of protein regulations in protein folding and oxidative stress proteins, they were absolutely different in regulation of photosynthesis and carbohydrate proteins. Even, tolerant mutant showed lower up regulation intensity in some oxidative stress proteins than wild-type parent. Protein–protein interaction (PPI) maps revealed core protein roles in regulating salt tolerance mechanisms in mutant lines and their wild-type parent. PPI maps were created with two algorithms including K-mean and MCL clusters to discover protein–protein interactions. By employing PPI, we found 4 core proteins including Peptidyl-prolyl cis–trans isomerase, 70 kDa heat shock protein, Ferredoxin and 2-Cys peroxiredoxin that were involved in protein folding and oxidative stress pathways. These core proteins can regulate a wide range of molecular processes like carbohydrates metabolism, photosynthesis and ATP synthesis. As a result, tolerant mutant would regulate salt tolerance due to efficient and ordered protein–protein interactions. Further studies are needed to illuminate the importance of ordered protein–protein interactions leading to salt tolerance. [ABSTRACT FROM AUTHOR]

Rationale: This study used proteomics‐based data‐independent acquisition (DIA) technology with the aim of screening for differential expression proteins in type I gastric neuroendocrine neoplasm (g‐NEN). Methods: Differential expression proteins in type I g‐NEN and peritumoral tissues were screened using DIA with liquid chromatography/tandem mass spectrometry (DIA‐LC/MS/MS). The identified proteins were then functionally analysed using bioinformatics methods. We selected the three most highly expressed proteins, combined with patients' clinical data, for prognostic analysis. Results: Compared with peritumoral tissues, 224 proteins were up‐regulated, and 70 were down‐regulated. The most significantly enriched biological processes and pathways were vacuolar proton‐transporting V‐type ATPase complex assembly and metabolism‐related pathways. PCSK1, FBXO2, ACSL1, IRS2, and PTPRZ1 expression was markedly up‐regulated in type I g‐NENs. High IRS2 expression significantly correlated with a shorter time to recurrence. Conclusions: Our study provides a comprehensive proteomic signature based on DIA‐LC/MS/MS and highlights high IRS2 expression as a potential prognostic marker for type I gNENs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Peanut peptides have good chelating ability with metal ions. However, there are few studies on the chelation mechanism of peanut peptides with calcium and absorption properties of peptide–calcium complex. RESULTS: Peptides with high calcium chelating rate were isolated and purified from peanut protein hydrolysate (PPH), and the chelation rate of component F21 was higher (81.4 ± 0.8%). Six peptides were identified from component F21 by liquid chromatography–tandem mass spectrometry, and the frequency of acidic amino acids and arginine in the amino acid sequence was higher in all six peptides. Peanut peptide–calcium complex (PPH21‐Ca) was prepared by selecting component F21 (PPH21). Ultraviolet analysis indicated that the chelate reaction occurred between peanut peptide and calcium ions. Fourier transform infrared analysis showed that the chelating sites were carboxyl and amino groups on the amino acid residues of peptides. Scanning electron microscopy revealed that the surface of peanut peptide had a smooth block structure, but the surface of the complex had a granular morphology. Caco‐2 cell model tests revealed that the bioavailability of PPH21‐Ca was 58.4 ± 0.5%, which was significantly higher than that of inorganic calcium at 37.0 ± 0.4%. CONCLUSION: Peanut peptides can chelate calcium ions by carboxyl and amino groups, and the peptide–calcium complex had higher bioavailability. This study provides a theoretical basis for the development of new calcium supplement products that are absorbed easily. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Paeoniae Radix Rubra (PRR) has been usedwidely to promote blood circulation and eliminate blood stasis in China clinical practice owing to its extensive pharmacological effects. However, the "quality markers" (Q-markers) of the antioxidant effects remains unknown. Object: To explore the Q-markers of antioxidant activity based on multiple strategies, which would provide reference for the quality evaluation of PRR based on specific pharmacodynamic-oriented. Methods: Firstly, the "fingerprint" profiles of 15 batches of PRR were acquired and identified by ultrahigh performance liquid chromatography-quadrupole time-of- flight tandem mass spectrometry (UHPLC-Q-TOF MS/MS) and the common peaks extracted. Meanwhile, the MTT assay was used to evaluate the effect of 15 batches of PRR on H2O2-induced oxidative stress in HT-22 cells. The antioxidant activity of PRR was investigated simultaneously by superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) commercial kits. The relationship between common peaks and antioxidant indexes were constructed by grey relational analysis (GRA) and partial least squares- discriminant analysis (PLS-DA) for the identification of preselected Q-markers. Secondly, experimental verification was conducted to investigate the protective effect of the preliminary components on HT-22 cells undergoing oxidative stress. Finally, for the further validation of effectiveness of antioxidant Q-markers, network pharmacology was applied to explore potential targets, and the molecular docking technology was used to value the binding ability of the potential active components of PRR to the antioxidant targets. Results: Thirty-seven common peaks from 15 batches of PRR were identified qualitatively by UHPLC-Q-TOF MS/MS. The MTT assay showed that PRR could reduce the oxidative damage induced by H2O2 upon HT-22 cells according to the index of MDA, SOD and GSH. Eight potential antioxidant components were screened by spectrum-effect correlation analysis: paeoniflorin, galloylpaeoniflorin, albiflorin, 1,2,3,4,6-o-pentagalloylglucose, benzoylpaeoniflorin, pinocembrin, oleanic acid, and isorhamnetin-3-o-nehesperidine. Each of these preliminary components showed significant protections on cellular oxidative stress (P < 0.05). Interleukin- 6 (IL-6), protein kinase B (AKT1), and tumor necrosis factor (TNF) were predicted to be the major potential targets of PRR, and the good binding ability were presented between the potential active components of PRR and each target as a whole. Conclusion: Eight components were identified as the antioxidant Q-markers of PRR based on an integrated multimodal strategy. [ABSTRACT FROM AUTHOR]

Cyclodepsipeptides (CDPs) represent a huge family of chemically and structurally diverse molecules with a wide ability for molecular interactions. CDPs are cyclic peptide‐related natural products made up of both proteinogenic and nonproteinogenic amino acids linked by amide and ester bonds. The combined use of different analytical methods is required to accurately determine their integral structures including stereochemistry, thus allowing deeper insights into their often‐intriguing bioactivities and their possible usefulness. Our goal is to present the various methods developed to accurately characterize CDPs. Presently, Marfey's method and NMR (nuclear magnetic resonance) are still considered the best for characterizing CDP configuration. Nevertheless, electrospray‐high resolution tandem mass spectrometry (ESI‐HRMS/MS) is of great value for efficiently resolving CDP's composition and sequences. For instance, recent data shows that the fragmentation of cationized CDPs (e.g., [M + Li]+ and [M + Na]+) leads to selective cleavage of ester bonds and specific cationized product ions (b series) useful to get unprecedented sequence information. Thus, after a brief presentation of their structure, biological functions, and biosynthesis, we also provide a historic overview of these various analytical approaches as well as their advantages and limitations with a special emphasis on the emergence of methods based on HRMS/MS through recent fundamental works and applications. [ABSTRACT FROM AUTHOR]

Background and aims: Aberrations in the immunoglobulin heavy chain (IgH) locus are associated with poor prognosis in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) patients. The primary objective of this pilot study is to enhance our understanding of the IgH phenotype by exploring the intracellular chiral metabolome. Materials and methods: Leukemia cells were isolated from the bone marrow of BCP-ALL pediatric patients at diagnosis. The samples' metabolome and transcriptome were characterized using untargeted chiral metabolomic and next-generation sequencing transcriptomic analyses. Results: For the first time D-amino acids were identified in the leukemic cells' intracellular metabolome from the bone marrow niche. Chiral metabolic signatures at diagnosis was indicative of a resistant phenotype. Through integrated network analysis and Pearson correlation, confirmation was obtained regarding the association of the IgH phenotype with several genes linked to poor prognosis. Conclusion: The findings of this study have contributed to the understanding that the chiral metabolome plays a role in the poor prognosis observed in an exceptionally rare patient cohort. The findings include elevated D-amino acid incorporation in the IgH group, the emergence of several unknown, potentially enantiomeric, metabolites, and insights into metabolic pathways that all warrant further exploration. [ABSTRACT FROM AUTHOR]

Clinical expression of coronavirus disease 2019 (COVID‐19) infectionis widely variable including fatal cases and patients with mild symptoms and a rapid resolution. We studied saliva from 63 hospitalized COVID‐19 patients and from 30 healthy controls by integrating large‐scale proteomics, peptidomics and targeted metabolomics to assess the biochemical alterations following the infection and to obtain a set of putative biomarkers useful for noninvasive diagnosis. We used an untargeted approach by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for proteomics and peptidomics analysis and targeted LC–multiple reaction monitoring/MS for the analysis of amino acids. The levels of 77 proteins were significantly different in COVID‐19 patients. Among these, seven proteins were found only in saliva from patients with COVID‐19, four were up‐regulated and three were down‐regulated at least five‐folds in saliva from COVID‐19 patients in comparison to controls. The analysis of proteins revealed a complex balance between pro‐inflammatory and anti‐inflammatory proteins and a reduced amount of several proteins with immune activity that possibly favours the spreading of the virus. Such reduction could be related to the enhanced activity of endopeptidases induced by the infection that in turn caused an altered balance of free peptides. In fact, on a total of 28 peptides, 22 (80%) were differently expressed in severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and control subjects. The multivariate analysis of such peptides permits to obtain a diagnostic algorithm that discriminate the two populations with a high diagnostic efficiency. Among amino acids, only threonine resulted significantly different between COVID‐19 patients and controls, while alanine levels were significantly different between COVID‐19 patients with different severity. In conclusion, the present study defined a set of molecules to be detected with a quick and easy method based on mass spectrometry tandem useful to reveal biochemical alterations involved in the pathogenesis of such a complex disease. Data are available via ProteomeXchange with identifier PXD045612. [ABSTRACT FROM AUTHOR]

Triphenylamine (TPA) and N‐heterotriangulene (N‐HTA) scaffolds with up to three oligophenyl extensions are investigated by electrospray ionization (tandem) mass spectrometry (ESI‐[MS/]MS). Due to their low oxidation potentials, all molecules readily form radical cations in the electrospray process. The energy‐resolved collision‐induced dissociation behaviour of the molecular ions is contrasted to that of the silver(I) adducts. Complexation with Ag(I) leads to the expected [1:1] and [2:1] complexes (MAg+ and M2Ag+); however, even [1:2] complexes (MAg22+) can be detected for molecules with two and three large π‐expansions to allow stabilization of two charges. The TPA scaffolds decompose only at high collision energies through the loss of peripheral tert‐butyl groups. A general mechanism for this is proposed commencing with a methyl loss and followed by the release of isobutene and butyl radical moieties. The N‐HTA‐based scaffolds are considerably less stable and molecular ions fragment at low collision energies. This is caused by the facile loss of methyl radicals from the dimethylmethylene‐bridged triangulene core. In contrast, complexation with Ag+ leads to a dramatic stabilization. Most interestingly, dissociation eventually proceeds via the loss of neutral AgCH3, which is indicative of strong bidentate, tweezer‐like bonding of Ag+ to the molecules. [ABSTRACT FROM AUTHOR]

Rationale: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Methods: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESIMS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. Results: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography- high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 µL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. Conclusions: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography- high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development. [ABSTRACT FROM AUTHOR]

Rationale: Illegal addition of anti-infective drugs to cosmetics at low concentrations has been found. The illicit addition of anti-infective drugs encompasses a wide variety of medications. The current sample purification methods are inadequate to detect all these compounds. A sensitive, wide-coverage, and weak-matrix-effect measurement method needs to be established to address this issue. Methods: Samples were extracted using acetonitrile, diluted 25 times, and then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect 111 anti-infective drugs. The method was validated and assessed for matrix effect before being applied to cosmetic products. Results: The calibration curves for the analytes exhibited a strong correlation coefficient (r > 0.995). The limit of detection ranged from 0.006 to 0.6 mg/kg. Matrix effects were significantly improved after a 25-fold dilution. The method was successfully applied to various cosmetics. Two of 82 samples tested contained lincomycin and miconazole, respectively. Conclusions: The developed method is quick and reliable to analyze anti-infective drugs in cosmetics, with potential for both qualitative and quantitative analyses. It is a valuable tool for cosmetic research and development, contributing to safer and more effective cosmetic products. [ABSTRACT FROM AUTHOR]