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1.
J Extracell Vesicles ; 11(2): e12184, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35119778

RESUMO

The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.


Assuntos
Vesículas Extracelulares , Proteômica , Vesículas Extracelulares/química , Humanos , Masculino , Espectrometria de Massas , Próstata , Proteômica/métodos , Ultracentrifugação
2.
J Transl Med ; 20(1): 199, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538547

RESUMO

BACKGROUND: Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. METHODS: 3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment. RESULTS: Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories. CONCLUSIONS: We demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.


Assuntos
Vesículas Extracelulares , Exposição à Radiação , Animais , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Espectrometria de Massas , Ratos , Ultracentrifugação
3.
Dis Markers ; 2022: 1758113, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35521635

RESUMO

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high mortality rate due to its poor diagnosis in the early stage. Here, we report a urinary metabolomic study on a cohort of CRC patients (n =67) and healthy controls (n =21) using ultraperformance liquid chromatography triple quadrupole mass spectrometry. Pathway analysis showed that a series of pathways that belong to amino acid metabolism, carbohydrate metabolism, and lipid metabolism were dysregulated, for instance the glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, glycolysis, and TCA cycle. A total of 48 differential metabolites were identified in CRC compared to controls. A panel of 12 biomarkers composed of chenodeoxycholic acid, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline were identified by Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis classification model and validated by Gradient Boosting (GB), Logistic Regression (LR), and Random Forest diagnostic model, which were able to discriminate CRC subjects from healthy controls. These urinary metabolic biomarkers provided a novel and promising molecular approach for the early diagnosis of CRC.


Assuntos
Neoplasias Colorretais , Biomarcadores/metabolismo , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Glicina , Humanos , Espectrometria de Massas/métodos , Metabolômica/métodos
4.
Methods Mol Biol ; 2477: 195-223, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524119

RESUMO

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to get a comprehensive picture of cellular behavior and to understand biological systems. In the past two decades, affinity purification coupled to mass spectrometry has become a powerful tool to comprehensively study interaction networks and their assemblies. To overcome initial limitations of the approach, in particular, the effect of protein and RNA degradation, loss of transient interactors, and poor overall yield of intact complexes from cell lysates, various modifications to affinity purification protocols have been devised over the years. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol as described here has been optimized for the yeast S. cerevisiae.


Assuntos
Proteínas , Saccharomyces cerevisiae , Cromatografia de Afinidade/métodos , Substâncias Macromoleculares/metabolismo , Espectrometria de Massas/métodos , Proteínas/química , Saccharomyces cerevisiae/genética
5.
Methods Mol Biol ; 2477: 225-236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524120

RESUMO

Multiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function, and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time, together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively nonabundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism.


Assuntos
Proteínas , Saccharomyces cerevisiae , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/metabolismo , Saccharomyces cerevisiae/genética
6.
Methods Enzymol ; 667: 303-338, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35525545

RESUMO

Dynamics of the protein kinase fold are deeply intertwined with its structure. The past three decades of kinase biophysical studies revealed key dynamic features of the kinase domain and, more recently, how these features may endow catalytically impaired kinases-or pseudokinases-with signaling properties. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is proving to be a valuable approach for studies of kinase and pseudokinase domain dynamics. Here, we briefly discuss the methods that have provided insights into protein kinase dynamics, describe how HDX-MS is being used to answer questions in the kinase/pseudokinase field, and provide a detailed protocol for collecting an HDX-MS dataset to study the impacts of small molecule binding to a pseudokinase domain. As more small molecules are discovered that can disrupt pseudokinase conformations, HDX-MS is likely to be a powerful approach for exploring drug-induced changes in pseudokinase dynamics and structure.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas Quinases
7.
Anal Chim Acta ; 1208: 339814, 2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35525585

RESUMO

Metabolism studies are one of the important steps in pharmaceutical research. LC-MS combined with metabolomics data-processing approaches have been developed for rapid screening of drug metabolites. Mass defect filter (MDF) is one of the LC/MS-based metabolomics data processing approaches and has been applied to screen drug metabolites. Although MDF can remove most interference ions from an incubation sample, the true positive rate of the retaining ions is relatively low (approximately 10%). To improve the efficacy of MDF, we developed a two-stage data-processing approach by combining MDF and stable isotope tracing (SIT) for metabolite identification. Pioglitazone (PIO), which is an antidiabetic drug used to treat type 2 diabetes mellitus, was taken as an example drug. Our results demonstrated that this new approach could substantially increase the validated rate from about 10% to 74%. Most of these validated metabolite signals (13/14) could be verified as PIO structure-related metabolites. In addition, we applied this approach to identify uncommon metabolite signals (a mass change beyond the window of 50 Da around its parent drug, MDF1). SIT could remove most interference ions (approximately 98%) identified by MDF1, and four out of five validated metabolite signals could be verified as PIO structure-related metabolites. Interestingly, a lot of the verified metabolites (10/17) were novel PIO metabolites. Among these novel metabolites, nine were thiazolidinedione ring-opening signals that might be related to the toxicity of PIO. Our developed approach could significantly improve the efficacy in drug metabolite identification compared with that of MDF.


Assuntos
Diabetes Mellitus Tipo 2 , Cromatografia Líquida/métodos , Humanos , Isótopos , Espectrometria de Massas/métodos , Metabolômica/métodos
8.
J Extracell Vesicles ; 11(5): e12213, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35524458

RESUMO

BACKGROUND: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. METHOD: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. RESULTS: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. CONCLUSION: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail.


Assuntos
Plaquetas , Vesículas Extracelulares , Ácido Edético/análise , Vesículas Extracelulares/química , Humanos , Espectrometria de Massas , Tetraspaninas/análise
9.
STAR Protoc ; 3(1): 101089, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35535160

RESUMO

Due to the low stoichiometry and highly transient nature of protein phosphorylation it is challenging to capture the dynamics and complexity of phosphorylation events on a systems level. Here, we present an optimized protocol to measure virus-induced phosphorylation events with high sensitivity using label free quantification-based phosphoproteomics. Specifically, we describe filter assisted protein digestion (FASP), enrichment of phosphopeptides, mass spectrometry, and subsequent bioinformatic analysis. For complete details on the use and execution of this protocol, please refer to Hunziker et al. (2022).


Assuntos
Fosfopeptídeos , Proteômica , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Fosforilação , Proteômica/métodos , Transdução de Sinais
10.
Methods Mol Biol ; 2469: 145-154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35508836

RESUMO

The localization of metabolites in plant tissues is often related to their biological function and biosynthesis. Mass spectrometry imaging (MSI) provides comprehensive information about the distribution of known and unknown compounds in tissues. In this protocol, we describe the use of laser desorption low-temperature plasma (LD-LTP) ionization MSI. This technology enables the direct analysis of native tissues under ambient conditions.


Assuntos
Lasers , Plantas , Temperatura Baixa , Espectrometria de Massas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Temperatura
11.
STAR Protoc ; 3(2): 101311, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35496788

RESUMO

Metabolites are crucial for bidirectional communication between host and microbiome. We describe a protocol for the isolation of organic and aqueous metabolites from mucosal scrapes and feces from mouse and human samples. Although some of the most reactive organic compounds may be lost, this approach generates a functionally reproducible metabolic extract containing both host and microbial compounds appropriate for quantitative mass spectrometry and functional characterization. Our mass spectrometry approach identifies low-abundant and difficult to identify microbially derived metabolites. For complete details on the use and execution of this protocol, please refer to Bell et al. (2021) and Das et al. (2020).


Assuntos
Microbiota , Animais , Fezes/química , Humanos , Espectrometria de Massas/métodos , Camundongos , Compostos Orgânicos/análise
12.
Front Immunol ; 13: 855976, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493528

RESUMO

Neoantigens are widely reported to induce T-cell response and lead to tumor regression, indicating a promising potential to immunotherapy. Previously, we constructed an open-access database, i.e., dbPepNeo, providing a systematic resource for human tumor neoantigens to storage and query. In order to expand data volume and application scope, we updated dbPepNeo to version 2.0 (http://www.biostatistics.online/dbPepNeo2). Here, we provide about 801 high-confidence (HC) neoantigens (increased by 170%) and 842,289 low-confidence (LC) HLA immunopeptidomes (increased by 107%). Notably, 55 class II HC neoantigens and 630 neoantigen-reactive T-cell receptor-ß (TCRß) sequences were firstly included. Besides, two new analytical tools are developed, DeepCNN-Ineo and BLASTdb. DeepCNN-Ineo predicts the immunogenicity of class I neoantigens, and BLASTdb performs local alignments to look for sequence similarities in dbPepNeo2.0. Meanwhile, the web features and interface have been greatly improved and enhanced.


Assuntos
Antígenos de Neoplasias , Neoplasias , Humanos , Espectrometria de Massas , Peptídeos , Receptores de Antígenos de Linfócitos T alfa-beta
13.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532271

RESUMO

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.


Assuntos
Proteômica , Análise de Célula Única , Animais , Linhagem da Célula , Espectrometria de Massas , Proteoma/metabolismo , Proteômica/métodos , Análise de Célula Única/métodos , Xenopus laevis/metabolismo
14.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532272

RESUMO

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model. One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS). ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.


Assuntos
Rutênio , Animais , Cisplatino/toxicidade , Larva , Espectrometria de Massas/métodos , Platina , Peixe-Zebra/fisiologia
15.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532275

RESUMO

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described-single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)-to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.


Assuntos
Aplysia , RNA , Animais , Aplysia/genética , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Neurônios/metabolismo , RNA/metabolismo , Processamento Pós-Transcricional do RNA
16.
Zhongguo Zhong Yao Za Zhi ; 47(7): 1765-1775, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-35534247

RESUMO

Based on the previous research results of our group and literature research, the chemical components, mechanisms, pharmacodynamics, and pharmacokinetics of Zingiberis Rhizoma Carbonisata were summarized to determine the quality markers(Q-markers) of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Our research group has clarified the differential components of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma, the meridian-warming hemostatic effect of Zingiberis Rhizoma Carbonisata, the related targets and pathways of the effect, the endogenous biomarkers of Zingiberis Rhizoma Carbonisata, and the hemodynamic processes of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Moreover, based on high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry(HPLC-DAD-ESIMS), a method for determining the content of Q-mar-kers was established. In conclusion, the study finally determined that gingerone, 6-shogaol, and diacetyl-6-gingerol were the Q-mar-kers of Zingiberis Rhizoma Carbonisata decoction pieces, and 6-gingerol, 8-gingerol, and 10-gingerol were Q-markers of Zingiberis Rhizoma decoction pieces. The result is expected to provide a reference for the establishment of quality standards for Zingiberis Rhizoma Carbonisata decoction pieces and Zingiberis Rhizoma decoction pieces.


Assuntos
Medicamentos de Ervas Chinesas , Rizoma , Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Gengibre , Espectrometria de Massas , Extratos Vegetais , Rizoma/química
17.
AAPS J ; 24(3): 66, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534647

RESUMO

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.


Assuntos
Bioensaio , Bioensaio/métodos , Biomarcadores/análise , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Padrões de Referência
18.
Artigo em Inglês | MEDLINE | ID: mdl-35537310

RESUMO

Traditional Chinese medicine (TCM) has been used in clinical settings for over 2000 years in China. The study of the absorption, distribution, metabolism, and excretion (ADME) of TCM in vivo could be beneficial for the discovery of the active components in TCM. However, the conventional strategies used for ADME research are based on rodent models and have the characteristics of lengthy experimental periods, complex processes, and extensive data processing, which make it difficult to perform rapid analyses and high-throughput ADME screening of the medicinal components of TCM. In this study, an integrated high-throughput research strategy for the in vivo ADME analysis of TCM was established based on a zebrafish model. Accordingly, a combination of ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS), desorption electrospray ionization-mass spectrometry (DESI-MS) imaging, and in-house non-targeted precise-and-thorough background-subtraction (PATBS) data post-processing techniques were successfully applied for the analysis of the metabolism of zebrafish exposed to Xiaoke pills. A total of 49 compounds related to Xiaoke pills (including 13 prototypical components and 36 metabolites) were detected in zebrafish. In total, 32 of them, including puerarin, daidzein, deoxyschizandrin, formononetin, and glibenclamide, which have been identified to have hypoglycemic activity in our previous studies and are phase I and phase II metabolites resulting from the hydroxylation, demethylation, glucuronidation, sulfation, and glycosylation of the prototypical components in vivo, were found in rats treated with Xiaoke pills. Furthermore, the overall distribution of the known compounds in zebrafish exposed to Xiaoke pills was explored using DESI-MS. In summary, this study provides a practical approach for the high-throughput screening of the active components of TCM using a zebrafish model.


Assuntos
Medicamentos de Ervas Chinesas , Peixe-Zebra , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Medicamentos de Ervas Chinesas/química , Espectrometria de Massas , Medicina Tradicional Chinesa/métodos , Ratos , Espectrometria de Massas por Ionização por Electrospray/métodos , Peixe-Zebra/metabolismo
19.
Sci Rep ; 12(1): 7360, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513691

RESUMO

Metabolomics approaches, such as direct analysis in real time-high resolution mass spectrometry (DART-HRMS), allow characterising many polar and non-polar compounds useful as authentication biomarkers of dairy chains. By using both a partial least squares discriminant analysis (PLS-DA) and a linear discriminant analysis (LDA), this study aimed to assess the capability of DART-HRMS, coupled with a low-level data fusion, discriminate among milk samples from lowland (silages vs. hay) and Alpine (grazing; APS) systems and identify the most informative biomarkers associated with the main dietary forage. As confirmed also by the LDA performed against the test set, DART-HRMS analysis provided an accurate discrimination of Alpine samples; meanwhile, there was a limited capacity to correctly recognise silage- vs. hay-milks. Supervised multivariate statistics followed by metabolomics hierarchical cluster analysis allowed extrapolating the most significant metabolites. Lowland milk was characterised by a pool of energetic compounds, ketoacid derivates, amines and organic acids. Seven informative DART-HRMS molecular features, mainly monoacylglycerols, could strongly explain the metabolomic variation of Alpine grazing milk and contributed to its classification. The misclassification between the two lowland groups confirmed that the intensive dairy systems would be characterised by a small variation in milk composition.


Assuntos
Leite , Silagem , Animais , Biomarcadores/análise , Dieta , Espectrometria de Massas , Leite/química , Silagem/análise
20.
Zhongguo Zhong Yao Za Zhi ; 47(8): 2121-2133, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-35531728

RESUMO

Based on the combination of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF) and Waters UNIFI software, the chemical constituents of the classic prescription Xiaochengqi Decoction were qualitatively analyzed and identified. The UPLC conditions are as follows: Acquity HSS T3 reverse phase column(2.1 mm ×100 mm, 1.8 µm), column temperature of 30 ℃, mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B), and flow rate of 0.3 mL·min~(-1). High-resolution MS data of Xiaochengqi Decoction were collected in ESI~(+/-) modes by Fast DDA. The structures of the chemical constituents were tentatively characterized or identified by UNIFI software according to the retention time of reference standards and characteristic fragment ions in MS profile, and literature data. A total of 233 components in Xiaochengqi Decoction were identified, with 93 from wine-processed Rhei Radix et Rhizoma, 104 from bran-processed Aurantii Fructus Immaturus, and 36 from ginger-processed Magnoliae Officinalis Cortex. These 233 components included anthraquinones, flavonoids, lignans, alkaloids, coumarins, and phenylethanoid glycosides. The result provided experimental evidence for the further study on establishment of quality standard and product development of the formula.


Assuntos
Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão/métodos , DDT/análogos & derivados , Medicamentos de Ervas Chinesas/química , Espectrometria de Massas , Rizoma/química , Software
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