RESUMEN
As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.
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Inmunidad Innata , Proteómica , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Western Blotting/métodos , Espectrometría de Masas/métodos , Inmunoprecipitación/métodos , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.
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Inmunoprecipitación , Fosfoproteínas , Espectrometría de Masas en Tándem , Fosforilación , Espectrometría de Masas en Tándem/métodos , Inmunoprecipitación/métodos , Cromatografía Liquida/métodos , Humanos , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Espectrometría de Masas/métodosRESUMEN
BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.
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Analgésicos Opioides , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Analgésicos Opioides/orina , Cromatografía Liquida/métodos , Factores de TiempoRESUMEN
Polylactic acid (PLA) straws hold eco-friendly potential; however, residual diisocyanates used to enhance the mechanical strength can generate carcinogenic primary aromatic amines (PAAs), posing health risks. Herein, we present a rapid, comprehensive strategy to detecting PAAs in 18 brands of food-grade PLA straws and assessing their migration into diverse food simulants. Surface-enhanced Raman spectroscopy was conducted to rapidly screen straws for PAAs. Subsequently, qualitative determination of migrating PAAs into various food simulants (4 % acetic acid, 10 % ethanol, 50 % ethanol) occurred at 70 °C for 2 h using liquid chromatography-mass spectrometry. Three PAAs including 4,4'-methylenedianiline, 2,4'-methylenedianiline, and 2,4-diaminotoluene were detected in all straws. Specifically, 2,4-diaminotoluene in 50 % ethanol exceeded specific migration limit of 2 µg/kg, raising safety concerns. Notably, PAAs migration to 10 % and 50 % ethanol surpassed that to 4 % acetic acid within a short 2-hour period. Moreover, PLA straws underwent varying degrees of shape changes before and after migration. Straws with poly(butylene succinate) resisted deformation compared to those without, indicating enhanced heat resistance, while poly(butyleneadipate-co-terephthalate) improved hydrolysis resistance. Importantly, swelling study unveiled swelling effect wasn't the primary factor contributing to the increased PAAs migration in ethanol food simulant, as there was no significant disparity in swelling degrees across different food simulants. FT-IR and DSC analysis revealed higher PAAs content in 50 % ethanol were due to highly concentrated polar ethanol disrupting hydrogen bonds and van der Waal forces holding PLA molecules together. Overall, minimizing contact between PLA straws and alcoholic foods is crucial to avoid potential safety risks posed by PAAs.
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Aminas , Poliésteres , Espectrometría Raman , Poliésteres/química , Espectrometría Raman/métodos , Cromatografía Liquida/métodos , Aminas/análisis , Aminas/química , Espectrometría de Masas/métodos , Contaminación de Alimentos/análisis , Embalaje de Alimentos , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Toxic cyanobacterial blooms impose a health risk to recreational users, and monitoring of cyanobacteria and associated toxins is required to assess this risk. Traditionally, monitoring for risk assessment is based on cyanobacterial biomass, which assumes that all cyanobacteria potentially produce toxins. While these methods may be cost effective, relatively fast, and more widely accessible, they often lead to an overestimation of the health risk induced by cyanotoxins. Monitoring methods that more directly target toxins, or toxin producing genes, may provide a better risk assessment, yet these methods may be more costly, usually take longer, or are not widely accessible. In this study, we compared six monitoring methods (fluorometry, microscopy, qPCR of 16S and mcyE, ELISA assays, and LC-MS/MS), of which the last three focussed on the most abundant cyanotoxin microcystins, across 11 lakes in the Netherlands during the bathing water season (May-October) of 2019. Results of all monitoring methods significantly correlated with LC-MS/MS obtained microcystin levels (the assumed 'golden standard'), with stronger correlations for methods targeting microcystins (ELISA) and microcystin genes (mcyE). The estimated risk levels differed substantially between methods, with 78 % and 56 % of alert level exceedances in the total number of collected samples for fluorometry and microscopy-based methods, respectively, while this was only 16 % and 6 % when the risk assessment was based on ELISA and LC-MS/MS obtained toxin concentrations, respectively. Integrating our results with earlier findings confirmed a strong association between microcystin concentration and the biovolume of potential microcystin-producing genera. Moreover, using an extended database consisting of 4265 observations from 461 locations across the Netherlands in the bathing water seasons of 2015 - 2019, we showed a strong association between fluorescence and the biovolume of potentially toxin-producing genera. Our results indicate that a two-tiered approach may be an effective risk assessment strategy, with first a biomass-based method (fluorometry, biovolume) until the first alert level is exceeded, after which the risk level can be confirmed or adjusted based on follow-up toxin or toxin gene analyses.
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Cianobacterias , Monitoreo del Ambiente , Floraciones de Algas Nocivas , Lagos , Microcistinas , Medición de Riesgo , Monitoreo del Ambiente/métodos , Microcistinas/análisis , Lagos/microbiología , Lagos/química , Países Bajos , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
To meet the needs of developing efficient extractive materials alongside the evolution of miniaturized sorbent-based sample preparation techniques, a mesoporous structure of g-C3N4 doped with sulfur as a heteroatom was achieved utilizing a bubble template approach while avoiding the severe conditions of other methods. In an effort to increase the number of adsorption sites, the resultant exfoliated structure was then modified with thymol-coumarin NADES as a natural sorbent modifier, followed by introduction into a nylon 6 polymer via an electrospinning process. X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and Brunauer-Emmett-Teller (BET) surface area analysis validated S-doped g-C3N4 and composite production. The prepared electrospun fiber nanocomposite, entailing satisfactory processability, was then successfully utilized as a sorbent in on-chip thin film micro-solid-phase extraction of non-steroidal anti-inflammatory drugs (NSAIDs) from saliva samples prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Utilizing a chip device, a thin film µ-SPE coupled with LC-MS/MS analysis yielded promising outcomes with reduced sample solution and organic solvents while extending lifetime of a thin film sorbent. The DES-modified S-doped g-C3N4 amount in electrospun was optimized, along with adsorption and desorption variables. Under optimal conditions, selected NSAIDs were found to have a linear range of 0.05-100.0 ng mL-1 with an R2 ≥ 0.997. The detection limits were ranged between 0.02 and 0.2 ng mL-1. The intra-day and inter-day precisions obtained were less than 6.0%. Relative recoveries were between 93.3 and 111.4%.
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Antiinflamatorios no Esteroideos , Disolventes Eutécticos Profundos , Grafito , Límite de Detección , Nanofibras , Saliva , Espectrometría de Masas en Tándem , Saliva/química , Espectrometría de Masas en Tándem/métodos , Grafito/química , Nanofibras/química , Humanos , Adsorción , Antiinflamatorios no Esteroideos/análisis , Porosidad , Disolventes Eutécticos Profundos/química , Cromatografía Liquida/métodos , Compuestos de Nitrógeno/química , Microextracción en Fase Sólida/métodos , Extracción en Fase Sólida/métodosRESUMEN
The fate of Alternaria toxin tenuazonic acid (TeA) during the processing chain of wheat flour products was systemically evaluated. TeA was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) in wheat grains and the corresponding wheat flour products produced throughout the whole chain. The results indicated that TeA contamination in wheat grains largely determines the level of TeA toxin present in byproducts, semi-finished products, and finished products of the processing of four types of simulated processed wheat flour products (e.g., dry noodles, steamed breads, baked breads, and biscuits). The different food processing techniques had different effects on the fate of TeA. Wheat flour processing can reduce the TeA content in wheat grains by 58.7-83.2 %, indicating that wheat flour processing is a key step in reducing the TeA content in the food chain. Among the four types of wheat flour products, the decreases in TeA content in biscuits (69.8-76.7 %) were greater than those in dry noodles (15.5-22.3 %) and steamed breads (24.9-43.6 %). In addition, the decreasing effect of TeA was especially obvious in the wheat flour product chain with a high level of contamination. The processing factors (PFs) for TeA were as low as 0.20 for the four wheat processing methods and as high as 1.24 for the dry noodle processing method. At the average and 95th percentiles, dietary exposure to TeA in Chinese consumers including infants and young children did not exceed the relevant threshold value of toxicological concern (TTC) of TeA (1.5 µg/kg body weight per day), indicating an acceptable health risk for Chinese consumers via wheat flour products. These findings provide new insight into the fate of TeA in the food chain and mycotoxin control on the safety of wheat flour products and public health.
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Alternaria , Harina , Contaminación de Alimentos , Manipulación de Alimentos , Espectrometría de Masas en Tándem , Ácido Tenuazónico , Triticum , Ácido Tenuazónico/análisis , Harina/análisis , Triticum/química , Contaminación de Alimentos/prevención & control , Contaminación de Alimentos/análisis , Manipulación de Alimentos/métodos , Micotoxinas/análisis , Humanos , Cromatografía Liquida , Pan/análisisRESUMEN
Donepezil (DPZ), a piperidine-based reversible cholinesterase inhibitor, finds extensive use in treating Alzheimer's disease (AD). Originally designed as an oral formulation, DPZ encounters drawbacks such as a brief duration of action and reduced treatment effectiveness in elderly patients with memory impairment or difficulty swallowing medications. To address these issues and improve patient compliance, researchers are actively exploring alternative DPZ formulations. Consequently, reliable methods are necessary to quantitate DPZ in biological samples for in vivo assessment. Therefore, we propose an efficient, sensitive, wide-dynamic, and cost-effective method for quantitating DPZ in rat plasma. Our method employs liquid-liquid extraction (LLE) followed by liquid chromatography and tandem mass spectrometry, enabling in vivo evaluation of novel DPZ formulations. Notably, our method requires only 20 µL of rat plasma and employs icopezil as the internal standard-a cost-effective compound with chemical similarity to DPZ. We meticulously optimized LLE conditions, taking into account factor interactions through design of experiments (DOE). Our rapid and straightforward extraction and purification involved using 500 µL of pure methyl tert-butyl ether to extract DPZ from the sample within five minutes. The dynamic range of the method extends from 0.5 ng/mL to 1,000 ng/mL, demonstrating excellent sensitivity and suitability for pharmacokinetic studies across diverse DPZ formulations. Following the FDA guidelines, we rigorously validated the developed method, evaluating selectivity, linearity (with a coefficient of determination ≥0.9999), accuracy (ranging from 96.0% to 109.6%), precision (≤13.9%), matrix effect (92.2% to 103.8%), recovery (98.5% to 106.8%), the lower limit of quantitation (0.5 ng/mL), and stability. Finally, we effectively employed the validated method for the long-term pharmacokinetic assessment of a DPZ formulation. We expect that this approach will make a substantial contribution to the advancement of new DPZ formulations, ultimately benefiting individuals afflicted by AD.
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Donepezilo , Extracción Líquido-Líquido , Piperidinas , Espectrometría de Masas en Tándem , Donepezilo/sangre , Donepezilo/farmacocinética , Animales , Espectrometría de Masas en Tándem/métodos , Extracción Líquido-Líquido/métodos , Ratas , Cromatografía Liquida/métodos , Piperidinas/sangre , Piperidinas/farmacocinética , Piperidinas/química , Inhibidores de la Colinesterasa/sangre , Inhibidores de la Colinesterasa/farmacocinética , Indanos/sangre , Indanos/farmacocinética , Masculino , Reproducibilidad de los Resultados , Ratas Sprague-Dawley , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Partial remission (PR) occurs in only half of people with new-onset type 1 diabetes (T1D) and corresponds to a transient period characterized by low daily insulin needs, low glycemic fluctuations and increased endogenous insulin secretion. While identification of people with newly-onset T1D and significant residual beta-cell function may foster patient-specific interventions, reliable predictive biomarkers of PR occurrence currently lack. We analyzed the plasma of children with new-onset T1D to identify biomarkers present at diagnosis that predicted PR at 3 months post-diagnosis. We first performed an extensive shotgun proteomic analysis using Liquid Chromatography-Tandem-Mass-Spectrometry (LCMS/MS) on the plasma of 16 children with new-onset T1D and quantified 98 proteins significantly correlating with Insulin-Dose Adjusted glycated hemoglobin A1c score (IDAA1C). We next applied a series of both qualitative and statistical filters and selected protein candidates that were associated to pathophysiological mechanisms related to T1D. Finally, we translationally verified several of the candidates using single-shot targeted proteomic (PRM method) on raw plasma. Taken together, we identified plasma biomarkers present at diagnosis that may predict the occurrence of PR in a single mass-spectrometry run. We believe that the identification of new predictive biomarkers of PR and ß-cell function is key to stratify people with new-onset T1D for ß-cell preservation therapies.
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Biomarcadores , Diabetes Mellitus Tipo 1 , Proteómica , Humanos , Diabetes Mellitus Tipo 1/sangre , Biomarcadores/sangre , Niño , Proteómica/métodos , Masculino , Femenino , Adolescente , Preescolar , Espectrometría de Masas en Tándem , Hemoglobina Glucada/análisis , Hemoglobina Glucada/metabolismo , Cromatografía Liquida , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Inducción de Remisión , Insulina/sangreRESUMEN
RATIONALE: Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. METHODS: This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. RESULTS: The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. CONCLUSIONS: These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.
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Anabolizantes , Camelus , Doping en los Deportes , Hígado , Detección de Abuso de Sustancias , Animales , Doping en los Deportes/prevención & control , Detección de Abuso de Sustancias/métodos , Hígado/metabolismo , Hígado/química , Anabolizantes/análisis , Anabolizantes/metabolismo , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Metandrostenolona/metabolismo , Metandrostenolona/análisis , Metandrostenolona/química , Cromatografía Líquida con Espectrometría de MasasRESUMEN
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.
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Biomarcadores , COVID-19 , Metabolómica , Proteómica , SARS-CoV-2 , Saliva , Espectrometría de Masas en Tándem , Humanos , COVID-19/virología , COVID-19/metabolismo , Saliva/química , Saliva/virología , Espectrometría de Masas en Tándem/métodos , Masculino , Femenino , Proteómica/métodos , Persona de Mediana Edad , Metabolómica/métodos , Biomarcadores/análisis , Biomarcadores/metabolismo , Adulto , Anciano , Cromatografía Liquida/métodos , Estudios de Casos y Controles , Proteoma/análisis , Proteoma/metabolismoRESUMEN
RATIONALE: Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. METHODS: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. RESULTS: Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. CONCLUSIONS: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.
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Elastina , Escherichia coli , Péptidos , Elastina/química , Escherichia coli/química , Péptidos/química , Péptidos/análisis , Concentración de Iones de Hidrógeno , Espectrometría de Masa por Ionización de Electrospray/métodos , Frío , Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Polipéptidos Similares a ElastinaRESUMEN
Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.
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Anticuerpos Monoclonales , Dependovirus , Vectores Genéticos , Dependovirus/genética , Dependovirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Afinidad de Anticuerpos/inmunología , AnimalesRESUMEN
Background: Plant-derived drugs are often preferred over synthetic drugs because of their superior safety profiles. Phenolic compounds and flavonoids-major plant components-possess antioxidant properties. Limited research has been conducted on the bioactive compounds and biochemical properties of Bellevalia pseudolongipes (Asparagaceae), an important pharmacological species endemic to Turkey. Therefore, the chemical composition and antioxidant properties of B. pseudolongipes were investigated in this study. Methods: The chemical composition of B. pseudolongipes was analyzed using liquid chromatography-high-resolution mass spectrometry, and radical scavenging and antioxidant activities were evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) tests. Results: Thirty-eight compounds were identified, including trans-cinnamic acid, caffeic acid, vitexin, schaftoside, orientin, and narirutin. B. pseudolongipes showed high antioxidant activity in antioxidant activity tests. Conclusion: These findings provide novel insights into the potential utility of B. pseudolongipes in the pharmaceutical, food, and cosmetics industries, highlighted by its significant antioxidant capacity.
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Antioxidantes , Espectrometría de Masas , Fitoquímicos , Extractos Vegetales , Antioxidantes/farmacología , Antioxidantes/química , Antioxidantes/análisis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Fitoquímicos/química , Fitoquímicos/análisis , Fitoquímicos/farmacología , Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Flavonoides/análisis , Flavonoides/química , Turquía , Fenoles/análisis , Fenoles/química , Ácidos Sulfónicos/química , Ácidos Sulfónicos/antagonistas & inhibidores , Compuestos de Bifenilo/química , BenzotiazolesRESUMEN
Vitamin A plays a critical role in various biological functions, including vision, cellular differentiation, and immune regulation. However, accurately assessing its status, particularly in obese individuals, presents challenges due to potential alterations in metabolism and distribution. This study utilized Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) methodology to precisely measure serum vitamin A concentrations in population of UAE. The methodology's reliability and precision, as demonstrated through validation procedures, underscore its potential utility in clinical settings. Employing the Multiple Reaction Monitoring mode of positive ion electrospray ionization, the LC-MS/MS system achieves a limit of detection (LOD) of 0.48 ng/mL in serum, while adhering to FDA-US regulations for accuracy and compliance. A key aspect of this study was the application of LC-MS/MS to assess vitamin A status in an obese population within UAE. By employing a diverse cohort of 452 Emirati participants, including 277 individuals from a randomized controlled trial who were assessed at baseline and at 6th month, and 175 healthy individuals aged 18-82 assessed at baseline, this study explores the relationship between obesity and vitamin A levels, shedding light on potential implications for health and well-being. It was an observational study based on a new vitamin A method and participants were asked to eat vitamin A rich foods. The robust performance of the LC-MS/MS methodology positions it as a valuable tool for clinical research. By accurately quantifying vitamin A levels in human serum, this methodology opens avenues for advancing our understanding of vitamin A physiology and its implications for health, particularly in obese populations. In summary, this LC-MS/MS methodology presents a potent tool for clinical studies, providing reliable, specific, and robust detection of vitamin A in human serum, thus, opening a new frontier for advancing our understanding of vitamin A related physiology and health in the obese population.
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Obesidad , Espectrometría de Masas en Tándem , Vitamina A , Humanos , Vitamina A/sangre , Obesidad/sangre , Adulto , Emiratos Árabes Unidos/epidemiología , Masculino , Persona de Mediana Edad , Espectrometría de Masas en Tándem/métodos , Femenino , Cromatografía Liquida/métodos , Anciano , Adolescente , Adulto Joven , Anciano de 80 o más Años , Reproducibilidad de los ResultadosRESUMEN
Assessment of personal formaldehyde (FA) exposure is most commonly carried out using formate as a biomarker, as it is the major product from FA metabolism. However, formate could also have originated from the metabolism of other endogenous and exogenous substances or from dietary intake, which may give rise to overestimated results with regard to FA exposure. We have developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an isotope-dilution method for rigorous quantitation of two major urinary FA conjugation products: thioproline (SPro) and thioprolinyl glycine (SPro-Gly), formed in the reaction between FA and endogenous cysteine or cysteinyl glycine, respectively, as marker molecules to assess personal FA exposure. Using this newly developed method, we measured the FA exposure levels in cigarette smokers, occupants of a chemistry research laboratory and typical domestic household, and visitors to a Chinese temple with a Pearson correlation coefficient greater than 0.94, showing a strong linear correlation between urinary adduct levels and the airborne FA level. It is believed that quantitation of urinary SPro and SPro-Gly may represent a noninvasive, interference-free method for assessing personal FA exposure.
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Biomarcadores , Formaldehído , Humanos , Biomarcadores/orina , Formaldehído/orina , Espectrometría de Masas en Tándem , Cromatografía Liquida , Glicina/análogos & derivados , Glicina/orina , Exposición a Riesgos Ambientales , Dipéptidos/orina , Tiazolidinas/orinaRESUMEN
Polymer conjugation has risen in importance over the past three decades as a means of increasing the in vivo half-life of biotherapeutics, with benefits including better stability, greater drug efficacy, and lower toxicity. However, the intrinsic variability of polymer synthesis results in products with broad distributions in chain length and branching structure, complicating quality control for successful functionalization and downstream conjugation. Frequently, a combination of several analytical techniques is required for comprehensive characterization. While liquid chromatography-mass spectrometry (LC-MS) is a powerful platform that can provide detailed molecular features of polymers, the mass spectra are inherently challenging to interpret due to high mass polydispersity and overlapping charge distributions. Here, by leveraging Fourier transform-based deconvolution and macromolecular mass defect analysis, we demonstrate a new way to streamline pharmaceutical polymer analysis, shedding light on polymer size, composition, branching, and end-group functionalization with the capability for reaction monitoring.
Asunto(s)
Análisis de Fourier , Espectrometría de Masas , Polímeros , Polímeros/química , Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Sustancias Macromoleculares/química , Peso Molecular , Cromatografía Líquida con Espectrometría de MasasRESUMEN
This study aimed to examine the metabolic profiles of Saccharomyces cerevisiae yeasts (WLS21 and Y41) in two phases of sparkling cider making (normal and pressure fermentation) by combining untargeted metabolomic with chemometrics. The results showed that of the 634 nonvolatile metabolites identified using LC-MS and 83 volatile metabolites identified by GC-MS, the differential metabolites were 226 and 54, respectively. Metabolic pathway and correlation analyses showed that aspartic acid, phenylalanine and tyrosine, glutamic acid and purine metabolism were associated with flavor formation. The pressure fermentation process increased apigenin, naringenin, toxifolin, pyridoxine and thiamine contents in the final cider. These findings provide useful information and new research ideas for the formation of flavor in sparkling cider and the regulation of phenolic and vitamin production by microbial stress fermentation.
Asunto(s)
Fermentación , Cromatografía de Gases y Espectrometría de Masas , Metabolómica , Saccharomyces cerevisiae , Metabolómica/métodos , Saccharomyces cerevisiae/metabolismo , Metaboloma , Bebidas Alcohólicas/análisis , Bebidas Alcohólicas/microbiología , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/metabolismo , Microbiología de Alimentos , Cromatografía Liquida/métodos , Redes y Vías MetabólicasRESUMEN
Lupins, and other legumes, have attained international interest due to their reported remarkable health benefits. Currently, the seed coats are discarded as waste or animal feed. The research presented here summarizes the potential for incorporating the seed coats into 'whole grain' foods. We aimed to identify metabolites found in the seed coats of nine commercial Australian cultivars of lupin (Lupinus angustifolius and L. albus species), and to evaluate and compare their functional, nutritional, antioxidant, and antidiabetic properties, along with in silico exploration of mechanisms of action for selected identified secondary metabolites. The seed coats were found to contain 79 to 90% dietary fibers and substantial quantity of essential macrometals. LC-QTOF MS-based, untargeted bioactive metabolite profiling explored a total of 673 chemical entities, and identified 63 bioactive secondary metabolites including: biophenols, unsaturated fatty acids, triterpenoids, alkaloids, and dietary prebiotics (insoluble fibers). The seed coats from these nine cultivars show substantial antioxidant activity. The cultivars of L. angustifolius inhibit α-amylase and α-glucosidase significantly in vitro. Moreover, in silico docking and dynamic simulation along with ADME/T analysis suggest that quercetin 3-methyl ether and 8-C-methylquercetin 3-methyl ether as molecules, novel in lupin seed coats, are responsible for the α-amylase and α-glucosidase inhibition. The findings indicated that lupin seed coats might be beneficial food components, rather than be discarded as 'mill waste'.
Asunto(s)
Antioxidantes , Hipoglucemiantes , Lupinus , Semillas , Antioxidantes/análisis , Semillas/química , Lupinus/química , Hipoglucemiantes/análisis , Simulación por Computador , Fibras de la Dieta/análisis , Valor Nutritivo , Australia , alfa-Amilasas/metabolismo , alfa-Amilasas/antagonistas & inhibidores , Cromatografía Liquida/métodos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Simulación del Acoplamiento Molecular , Inhibidores de Glicósido Hidrolasas/farmacología , Espectrometría de Masas/métodosRESUMEN
BACKGROUND: TRY-NAD metabolic network includes TRY (tryptophan), 5-HT (5-hydroxytryptamine), KYN (kynurenine), and NAD (nicotinamide adenine dinucleotide) pathway, which plays a significant role in neurological diseases and ageing. It is important to monitor these metabolites for studying the pathological anatomy of disease and treatment of responses evaluation. Although previous studies have reported quantitative methods for several metabolites in the network, the bottlenecks of simultaneously quantifying the whole metabolic network are their similar structures, diverse physico-chemical properties, and instability. Standardized protocols for the whole metabolic network are still missing, which hinders the in-depth study of TRY-NAD metabolic network in laboratory research and clinical screening. RESULTS: We developed a LC-MS/MS method for quantifying 28 metabolites in the TRY-NAD network simultaneously. Optimization was done for the mass spectral parameters, chromatographic conditions and sample pretreatment process. The developed method was fully validated in terms of standard curves, sensitivity, carryover, recovery, matrix effect, accuracy, precision, and stability. The pretreatment of 30 samples only takes 90 min, and the LC-MS/MS running time of one sample is only 13 min. With this method, we bring to light the chaos of global TRY-NAD metabolic network in sleep deprivation mice for the first time, including serum, clotted blood cells, hippocampus, cerebral cortex, and liver. NAD pathway levels in brain and blood decreased, whereas the opposite happened in the liver. The 5-HT pathway decreased and the concentration of KYN increased significantly in the brain. The concentration of many metabolites in KYN pathway (NAD+ de novo synthesis pathway) increased in the liver. SIGNIFICANCE: This method is the first time to determine the metabolites of KYN, 5-HT and NAD pathway at the same time, and it is found that TRY-NAD metabolic network will be disordered after sleep deprivation. This work clarifies the importance of the pH of the extraction solution, the time and temperature control in pretreatment in standardized protocols building, and overcoming the problems of inconsistent sample pretreatment, separation, matrix effect interference and potential metabolite degradation. This method exhibits great prospects in providing more information on metabolic disturbances caused by sleep deprivation as well as neurological diseases and ageing.