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Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.
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Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Mapeo de Interacción de Proteínas , Factores de Transcripción , Mapeo de Interacción de Proteínas/métodos , Humanos , Factores de Transcripción/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Unión Proteica , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Endonucleasas , Enzimas MultifuncionalesRESUMEN
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
Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.
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Inmunidad Innata , Lisina , Humanos , Acetilación , Lisina/metabolismo , Células HEK293 , Inmunoprecipitación/métodos , Macrófagos/inmunología , Macrófagos/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Animales , Espectrometría de Masas/métodos , Ratones , Fibroblastos/metabolismo , Fibroblastos/inmunología , Fibroblastos/virologíaRESUMEN
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
Perilla leaf oil (PLO) is a global premium vegetable oil with abundant nutrients and substantial economic value, rendering it susceptible to potential adulteration by unscrupulous entrepreneurs. The addition of cinnamon oil (CO) is one of the main adulteration avenues for illegal PLOs. In this study, new and real-time ambient mass spectrometric methods were developed to detect CO adulteration in PLO. First, atmospheric solids analysis probe tandem mass spectrometry combined with principal component analysis and principal component analysis-linear discriminant analysis was employed to differentiate between authentic and adulterated PLO. Then, a spectral library was established for the instantaneous matching of cinnamaldehyde in the samples. Finally, the results were verified using the SRM mode of ASAP-MS/MS. Within 3 min, the three methods successfully identified CO adulteration in PLO at concentrations as low as 5% v/v with 100% accuracy. The proposed strategy was successfully applied to the fraud detection of CO in PLO.
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Cinnamomum zeylanicum , Contaminación de Alimentos , Hojas de la Planta , Aceites de Plantas , Contaminación de Alimentos/análisis , Aceites de Plantas/química , Aceites de Plantas/análisis , Hojas de la Planta/química , Cinnamomum zeylanicum/química , Perilla/química , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas/métodosRESUMEN
Formic and acetic acids are the most abundant gaseous organic acids and play the key role in the atmospheric chemistry. In iodine-adduct chemical ionization mass spectrometry (CIMS), the low utilization efficiency of methyl iodide and humidity interference are two major issues of the vacuum ultraviolet (VUV) lamp initiated CIMS for on-line gaseous formic and acetic acids analysis. In this work, we present a new CIMS based on VUV lamp, and the ion-molecular reactor is separated into photoionization and chemical ionization zones by a reducer electrode. Acetone was added to the photoionization zone, and the VUV photoionization acetone provided low-energy electrons for methyl iodide to generate I-, and the addition of acetone reduced the amount of methyl iodide by 2/3. In the chemical ionization zone, a headspace vial containing ultrapure water was added for humidity calibration, and the vial changes the sensitivity as a function of humidity from ambiguity to well linear correlation (R2 > 0.95). With humidity calibration, the CIMS can quantitatively measure formic and acetic acids in the humidity range of 0%-88% RH. In this mode, limits of detection of 10 and 50 pptv are obtained for formic and acetic acids, respectively. And the relative standard deviation (RSD) of quantitation stability for 6 days were less than 10.5%. This CIMS was successfully used to determine the formic and acetic acids in the underground parking and ambient environment of the Shandong University campus (Qingdao, China). In addition, we developed a simple model based formic acid concentration to assess vehicular emissions.
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Espectrometría de Masas , Espectrometría de Masas/métodos , Contaminantes Atmosféricos/análisis , Yoduros/análisis , Yoduros/química , Rayos Ultravioleta , Formiatos/análisis , Formiatos/química , Atmósfera/química , Monitoreo del Ambiente/métodos , Procesos Fotoquímicos , Ácido Acético/análisis , Ácido Acético/química , Hidrocarburos Yodados/análisis , Hidrocarburos Yodados/químicaRESUMEN
Lake Baiyangdian is one of China's largest macrophyte - derived lakes, facing severe challenges related to water quality maintenance and eutrophication prevention. Dissolved organic matter (DOM) was a huge carbon pool and its abundance, property, and transformation played important roles in the biogeochemical cycle and energy flow in lake ecosystems. In this study, Lake Baiyangdian was divided into four distinct areas: Unartificial Area (UA), Village Area (VA), Tourism Area (TA), and Breeding Area (BA). We examined the diversity of DOM properties and sources across these functional areas. Our findings reveal that DOM in this lake is predominantly composed of protein - like substances, as determined by excitation - emission matrix and parallel factor analysis (EEM - PARAFAC). Notably, the exogenous tyrosine-like component C1 showed a stronger presence in VA and BA compared to UA and TA. Ultrahigh - resolution mass spectrometry (FT - ICR MS) unveiled a similar DOM molecular composition pattern across different functional areas due to the high relative abundances of lignan compounds, suggesting that macrophytes significantly influence the material structure of DOM. DOM properties exhibited specific associations with water quality indicators in various functional areas, as indicated by the Mantel test. The connections between DOM properties and NO3N and NH3N were more pronounced in VA and BA than in UA and TA. Our results underscore the viability of using DOM as an indicator for more precise and scientific water quality management.
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Monitoreo del Ambiente , Lagos , Lagos/química , China , Monitoreo del Ambiente/métodos , Eutrofización , Sustancias Húmicas/análisis , Calidad del Agua , Espectrometría de Masas/métodos , Contaminantes Químicos del Agua/análisis , EcosistemaRESUMEN
BACKGROUND: Mass spectrometry (MS)-based proteomics is a powerful tool for identifying and quantifying proteins. However, chimeric spectra caused by the fragmentation of multiple precursors within the same isolation window impair the accuracy of peptide identification and isobaric mass tag-based quantification. While there have been advances in computational deconvolution of chimeric spectra and methods to further separate the peptides by ion mobility or through MSn, the use of narrower isolation windows to decrease the fraction of chimeric species remains to be fully explored. RESULTS: We present results obtained on a SCIEX TripleTOF instrument where the quadrupole was optimized and tuned for precursor isolation at 0.1 Da (FWHH). Using a three-proteome model (trypsin digest of protein lysates from yeast, human and E. coli) and 8-plex iTRAQ labeling to document the interference effect, we investigated the impact of co-fragmentation on spectral purity, identification accuracy and quantification accuracy. The narrow quadrupole isolation window significantly improved the spectral purity and reduced the interference of non-target precursors on quantification accuracy. The high-resolution isolation strategy also reduced the number of false identifications caused by chimeric spectra. While these improvements came at the cost of sensitivity loss, combining high-resolution isolation with other advanced techniques, including ion mobility, may result in improved accuracy in identification and quantification. SIGNIFICANCE: Compared to standard-resolution quadrupole isolation (0.7 Da), high-resolution quadrupole isolation (0.1 Da) significantly improved the spectral purity and quantification accuracy while reducing the number of potential false identifications caused by chimeric spectra, thus showing excellent potential for further development to analyze clinical proteomics samples, for which high accuracy is essential.
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Proteómica , Proteómica/métodos , Humanos , Iones/química , Escherichia coli/química , Saccharomyces cerevisiae/química , Péptidos/química , Péptidos/análisis , Espectrometría de Masas/métodosRESUMEN
Crosslinking mass spectrometry (MS) has emerged as an important technique for elucidating the in-solution structures of protein complexes and the topology of protein-protein interaction networks. However, the expanding user community lacked an integrated visualisation tool that helped them make use of the crosslinking data for investigating biological mechanisms. We addressed this need by developing xiVIEW, a web-based application designed to streamline crosslinking MS data analysis, which we present here. xiVIEW provides a user-friendly interface for accessing coordinated views of mass spectrometric data, network visualisation, annotations extracted from trusted repositories like UniProtKB, and available 3D structures. In accordance with recent recommendations from the crosslinking MS community, xiVIEW (i) provides a standards compliant parser to improve data integration and (ii) offers accessible visualisation tools. By promoting the adoption of standard file formats and providing a comprehensive visualisation platform, xiVIEW empowers both experimentalists and modellers alike to pursue their respective research interests. We anticipate that xiVIEW will advance crosslinking MS-inspired research, and facilitate broader and more effective investigations into complex biological systems.
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Reactivos de Enlaces Cruzados , Espectrometría de Masas , Espectrometría de Masas/métodos , Reactivos de Enlaces Cruzados/química , Programas Informáticos , Proteínas/química , Mapeo de Interacción de Proteínas/métodos , Bases de Datos de Proteínas , Interfaz Usuario-Computador , Mapas de Interacción de ProteínasRESUMEN
The platinum-based anticancer drug cisplatin and its analog carboplatin are the most used chemotherapeutic agents worldwide. It is estimated that approximately half of all cancer patients are treated with platinum drugs at some point during the therapy regimen. Cisplatin covalently binds to purine nucleobases to form DNA adducts. Cisplatin therapy is faced with two key challenges. First, despite the initial response, many patients develop cisplatin resistance. Reduced cellular accumulation of cisplatin is one common cause of therapy resistance. Second, cisplatin treatment causes general cytotoxicity, leading to severe side effects. Monitoring the subcellular concentration of platinum chemotherapeutics will help yield clinical efficacy with the minimum possible dose. Inductively coupled plasma-mass spectrometry (ICP-MS) is an analytical technique to quantify the elemental composition of various types of liquified bulk samples with high sensitivity. This article describes quantifying cisplatin accumulation in chromatin and total cell lysate using ICP-MS. The method involves treating cells with cisplatin, isolating RNA-free DNA, digesting samples, ICP-MS instrumentation, and data analysis. Although we describe these steps in one cancer cell line, the protocol can be adapted to any cell line or tissue. The protocol should be a valuable resource for investigators interested in accurate measurement of subcellular concentration of platinum and other metallo-drugs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cell culture conditions for A2780 cells and cisplatin treatment Basic Protocol 2: Isolating cellular fractions and sample quantitation Basic Protocol 3: Sample digestion, ICP-MS data collection, and analysis.
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Antineoplásicos , Cisplatino , Espectrometría de Masas , Humanos , Cisplatino/metabolismo , Cisplatino/farmacología , Espectrometría de Masas/métodos , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Platino (Metal)/química , Platino (Metal)/metabolismo , Línea Celular Tumoral , Cromatina/metabolismoRESUMEN
Canine reproduction differs from that of many other domestic animals, and increased knowledge on biochemical changes during canine pregnancy is important for investigations of infertility or subfertility. The total glycosylation pattern, i.e., the glycome, of body fluids reflects cellular status in health and disease. The aim of the present pilot study was to investigate pregnancy-related changes of the serum N-glycome in bitches. A method based on Rapifluor HILIC-UPLC-FLR-MS was optimized and applied for analysis and quantification of N-glycans in canine serum. Serum samples from six pregnant and five non-pregnant bitches, collected at four well-defined time points, were included. The levels of sialylated and galactosylated complex glycans were significantly elevated in serum from pregnant bitches, consistent with previous reports on human pregnancy. The levels of fucosylated and agalactosylated glycans decreased significantly in pregnant dogs. In non-pregnant dogs, the glycosylation pattern did not change during the cycle. Pregnancy is an inflammatory state, but our findings during canine pregnancy are quite the opposite to changes that have previously been described for dogs with a known parasitic infection. Evaluation of the canine glycome may thus be valuable in studies of canine pregnancy, possibly differing inflammatory changes related to pregnancy to those caused by an infection.
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Polisacáridos , Animales , Perros , Femenino , Glicosilación , Embarazo , Polisacáridos/sangre , Polisacáridos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Preñez/sangre , Espectrometría de Masas/métodos , Proyectos PilotoRESUMEN
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
RATIONALE: The oxygen stable isotope ratio (δ18O) of the sugar-rich fraction of fruit juice is important as a tracer of the geographical origin of raw material. This study sought to minimize the inter-day variation of δ18O attributable to the influence of water to accurately monitor geographical origin labeling. METHODS: Two drying devices (freeze dryer and vacuum oven) were compared. Then, two humidity levels (normal and low humidity) at which the samples were placed after drying were compared. The low-humidity environment was constructed using a glove bag and pure argon gas. δ18O was measured using thermal conversion elemental analyzer/isotope ratio mass spectrometry. Improvements were made to the measurement method based on aforementioned analyses results, and the performance of the initial and improved methods was compared. RESULTS: δ18O of juice dried in a vacuum oven was 3.30 lower than that of juice dried in a freeze dryer. Moreover, δ18O of juice samples exposed to normal humidity was 3.74 lower than that of samples exposed to low humidity. The combined inter-day and intra-day standard deviation was reduced from 1.20 in the initial method to 0.42 in the improved method. CONCLUSIONS: This study describes a pretreatment method for δ18O measurement in the sugar-rich fraction of fruit juice with less inter-day variation, and it will be useful for monitoring geographical origin labeling.
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Jugos de Frutas y Vegetales , Espectrometría de Masas , Isótopos de Oxígeno , Isótopos de Oxígeno/análisis , Jugos de Frutas y Vegetales/análisis , Espectrometría de Masas/métodos , Humedad , Azúcares/análisis , Azúcares/químicaRESUMEN
RATIONALE: Sulfur isotopes are increasingly used as mobility indicators in humans and animals in biology, archaeology, and forensics. However, there has been a lack of modern sulfur isotope baseline "isoscape" studies using modern plants and animals, largely due to the possibility of contamination of the S isotope values by modern pollution. METHODS: We collected plants from across a 900-km east-west transect of British Columbia Canada and measured their sulfur isotope values. We then used a random forest model to determine which variables best explained the isotope data patterning and produced a sulfur isoscape for the southern region of British Columbia. RESULTS: We see clear patterning in the plant sulfur isotope values that relate to geographical location and rainfall. Our model also shows that for this study area, it is unlikely that there is a significant influence of anthropogenic pollution on plant δ34S values. We also discuss the use of plants as a substrate for sulfur isoscapes and possible explanations for the often-observed difference between plant and animal δ34S values from the same region, related to differing sources of sulfur in plants compared to amino acids in human and animal tissues. CONCLUSIONS: We found that for areas of the world where sulfur pollution is likely less widespread, it is possible to produce a modern plant S isoscape that should be an accurate baseline for mobility studies. Using random forest modelling, we have produced a baseline sulfur isoscape map of southern British Columbia that can be used for ecology, forensic and archaeological studies.
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Plantas , Isótopos de Azufre , Isótopos de Azufre/análisis , Plantas/química , Colombia Británica , Humanos , Animales , Azufre/análisis , Azufre/química , Espectrometría de Masas/métodosRESUMEN
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
Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.
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Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Ratones , Humanos , Espectrometría de Masas/métodos , Mapeo de Interacción de ProteínasRESUMEN
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.
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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
Native mass spectrometry (MS) is widely employed to study the structures and assemblies of proteins ranging from small monomers to megadalton complexes. Molecular dynamics (MD) simulation is a useful complement as it provides the spatial detail that native MS cannot offer. However, MD simulations performed in the gas phase have suffered from rapidly increasing computational costs with the system size. The primary bottleneck is the calculation of electrostatic forces, which are effective over long distances and must be explicitly computed for each atom pair, precluding efficient use of methods traditionally used to accelerate condensed-phase simulations. As a result, MD simulations have been unable to match the capacity of MS in probing large multimeric protein complexes. Here, we apply the fast multipole method (FMM) for computing the electrostatic forces, recently implemented by Kohnke et al. (J. Chem. Theory Comput., 2020, 16, 6938-6949), showing that it significantly enhances the performance of gas-phase simulations of large proteins. We assess how to achieve adequate accuracy and optimal performance with FMM, finding that it expands the accessible size range and time scales dramatically. Additionally, we simulate a 460 kDa ferritin complex over microsecond time scales, alongside complementary ion mobility (IM)-MS experiments, uncovering conformational changes that are not apparent from the IM-MS data alone.