Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.424
Filtrar
1.
J Agric Food Chem ; 68(1): 409-417, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31833363

RESUMO

Naringin has been documented to possess various bioactivities. Due to thorny endogenous interferences, the metabolism pathways of naringin and exact amounts of derived phenolic catabolites have not been definitely assigned. In this work, stable isotope-labeling-based liquid chromatography-mass spectrometry methods were developed to eliminate the endogenous interferences. [2',3',5',6'-D4]-naringin was orally administrated to rats. Urine and feces samples were collected and then analyzed with ultrahigh-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS). A total of 21 flavonoid metabolites and 11 phenolic catabolites were screened. The metabolism and catabolism pathways were proposed. Furthermore, deuterated naringin and its main metabolites were determined with rapid resolution liquid chromatography tandem triple quadrupole mass spectrometry (RRLC-QqQ-MS/MS). The overall recovery of ingested deuterated naringin was calculated as 56.9% without endogenous interferences. The obtained results provide essential information for further pharmacological studies of naringin.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Fezes/química , Flavanonas/química , Flavanonas/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Animais , Medicamentos de Ervas Chinesas/metabolismo , Feminino , Flavanonas/urina , Masculino , Ratos , Ratos Sprague-Dawley
2.
Chem Commun (Camb) ; 56(5): 723-726, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31840690

RESUMO

A new prosthetic group is reported for 18F-labelling of peptides and proteins based on the chemoselective ligation of potassium acyltrifluoroborates (KATs) and hydroxylamines without any detectable 18F/19F isotope exchange at the acyltrifluoroborate moiety. The new building block is appended via a common amide bond at room temperature with no need for protecting groups which enables an effective orthogonal 18F-radiolabelling.


Assuntos
Boratos/química , Radioisótopos de Flúor/química , Marcação por Isótopo/métodos , Peptídeos/química , Proteínas/química , Piridinas/química , Animais , Indicadores e Reagentes/química , Masculino , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Proteínas/metabolismo , Compostos Radiofarmacêuticos/química , Temperatura Ambiente
3.
Nat Protoc ; 14(12): 3333-3365, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31685960

RESUMO

Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.


Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Marcação por Isótopo/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas/fisiologia , Proteínas/metabolismo , Proteólise , Proteoma/metabolismo , Reprodutibilidade dos Testes , Fluxo de Trabalho
4.
BMC Bioinformatics ; 20(1): 549, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694522

RESUMO

BACKGROUND: Mass spectra are usually acquired from the Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for isotope labeled proteomics experiments. In such experiments, the mass profiles of labeled (heavy) and unlabeled (light) peptide pairs are represented by isotope clusters (2D or 3D) that provide valuable information about the studied biological samples in different conditions. The core task of quality control in quantitative LC-MS experiment is to filter out low-quality peptides with questionable profiles. The commonly used methods for this problem are the classification approaches. However, the data imbalance problems in previous control methods are often ignored or mishandled. In this study, we introduced a quality control framework based on the extreme gradient boosting machine (XGBoost), and carefully addressed the imbalanced data problem in this framework. RESULTS: In the XGBoost based framework, we suggest the application of the Synthetic minority over-sampling technique (SMOTE) to re-balance data and use the balanced data to train the boosted trees as the classifier. Then the classifier is applied to other data for the peptide quality assessment. Experimental results show that our proposed framework increases the reliability of peptide heavy-light ratio estimation significantly. CONCLUSIONS: Our results indicate that this framework is a powerful method for the peptide quality assessment. For the feature extraction part, the extracted ion chromatogram (XIC) based features contribute to the peptide quality assessment. To solve the imbalanced data problem, SMOTE brings a much better classification performance. Finally, the XGBoost is capable for the peptide quality control. Overall, our proposed framework provides reliable results for the further proteomics studies.


Assuntos
Marcação por Isótopo/métodos , Marcação por Isótopo/normas , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Algoritmos , Área Sob a Curva , Humanos , Peptídeos/química , Peptídeos/metabolismo , Controle de Qualidade , Curva ROC , Reprodutibilidade dos Testes
5.
Artigo em Inglês | MEDLINE | ID: mdl-31670058

RESUMO

Human studies investigating the efficacy of emergency decontamination protocols for chemical incidents require the use of non-hazardous chemical simulants. Methyl salicylate (MeS) has almost exclusively been used for this purpose. Whilst MeS is a simulant of the chemical warfare agent (CWA) sulphur mustard, it is not an ideal simulant for many other chemical threats with greater persistence and lower volatility. Benzyl salicylate (BeS) has been investigated here as a low toxicity simulant for lower volatility, persistent chemical threat agents and toxic industrial chemicals (TICs). To evaluate the suitability of BeS as a simulant for human decontamination studies a gas chromatography-triple quadrupole mass spectrometry method was designed, optimised and validated, for the analysis of human skin and hair. Quantification was achieved using isotope-dilution, EI and collision-induced dissociation and multiple reaction monitoring for both qualifier and quantifier ion transitions. The mass transitions were m/z 285 → 91 and m/z 210 → 181, respectively for the quantifier and qualifier ions of BeS, and m/z 289 → 91 and m/z 214 → 185 for the quantifier and qualifier ions for the BeS-d4 internal standard, respectively. The method exhibited excellent coefficients of determination (R2 = 0.9992-0.9999) with LOD and LOQ values at 0.023 ng/ml and 0.23 ng/ml. Across three Quality Controls (QCs), 11.5 ng/ml, 115 ng/ml and 1150 ng/ml) average accuracy (intra-day 95.6-100.3%, inter-day 98.5-104.91%) and precision (intra-day RSD (%) 2-13.7%, inter-day RSD (%) 3.3-8.8%) were determined. The validated method was applied in a proof of principle volunteer study for the determination of total BeS recovered from skin and hair. The average total BeS recovery after 70 min was 37.9% from skin and there was a significant increase between baseline and post-intervention levels for hair. These data demonstrate that BeS is an appropriate simulant for persistent chemicals and that the analytical method employed here is suitable for BeS analysis in human studies.


Assuntos
Substâncias para a Guerra Química/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Salicilatos/análise , Espectrometria de Massas em Tandem/métodos , Adulto , Descontaminação/métodos , Feminino , Cabelo , Humanos , Marcação por Isótopo/métodos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Pele
6.
Environ Int ; 133(Pt B): 105235, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31675570

RESUMO

Pathogens are known to survive in compost and to regrow under the influence of certain factors, such as moisture content, temperature and nutrient availability. Dead biomass, by providing available nutrients, is a factor that may affect pathogen regrowth. However, the indigenous microorganisms, including pathogens, that grown on the dead biomass of compost have not yet been identified. Here, the regrowth potential of the pathogenic indicator bacterium Escherichia coli in the presence of dead bacterial biomass was determined, and the biomass metabolizers that grew competitively with E. coli were identified by high-sensitivity stable isotope probing of rRNA. Culture-dependent analysis indicated that the addition of dead bacterial biomass did not stimulate E. coli growth. High-throughput analysis of density-resolved 16S rRNA molecules from compost samples amended with carbon-13-labeled dead bacterial biomass revealed dead bacterial-assimilating bacteria, including Sphingobium sp., myxobacterial lineages and Bacillales. These bacteria are potentially competitive with pathogens due to their preferential assimilation of dead biomass in compost.


Assuntos
Bactérias/metabolismo , Biomassa , Marcação por Isótopo/métodos , Microbiologia do Solo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Compostagem , Escherichia coli/metabolismo , Consórcios Microbianos
7.
Nat Protoc ; 14(10): 2878-2899, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31515516

RESUMO

Engineered nanomaterials (NMs) are often compositionally indistinguishable from their natural counterparts, and thus their tracking in the environment or within the biota requires the development of appropriate labeling tools. Stable isotope labeling has become a well-established such tool, developed to assign 'ownership' or a 'source' to engineered NMs, enabling their tracing and quantification, especially in complex environments. A particular methodological challenge for stable isotope labeling is to ensure that the label is traceable in a range of environmental or biological scenarios but does not induce modification of the properties of the NM or lose its signal, thus retaining realism and relevance. This protocol describes a strategy for stable isotope labeling of several widely used metal and metal oxide NMs, namely ZnO, CuO, Ag, and TiO2, using isotopically enriched precursors, namely 67Zn or 68Zn metal, 65CuCl2, 107Ag or 109Ag metal, and 47TiO2 powder. A complete synthesis requires 1-8 d, depending on the type of NM, the precursors used, and the synthesis methods adopted. The physicochemical properties of the labeled particles are determined by optical, diffraction, and spectroscopic techniques for quality control. The procedures for tracing the labels in aquatic (snail and mussel) and terrestrial (earthworm) organisms and for monitoring the environmental transformation of labeled silver (Ag) NMs are also described. We envision that this labeling strategy will be adopted by industry to facilitate applications such as nanosafety assessments before NMs enter the market and environment, as well as for product authentication and tracking.


Assuntos
Marcação por Isótopo/métodos , Nanoestruturas/química , Animais , Organismos Aquáticos/química , Cobre/química , Monitoramento Ambiental/métodos , Isótopos/química , Oligoquetos/química , Reprodutibilidade dos Testes , Prata/química , Caramujos/química , Titânio/química , Óxido de Zinco/química
8.
Anal Chim Acta ; 1082: 106-115, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472699

RESUMO

Sphingoid bases (SBs) are one of important components of cell membranes, playing important roles in cellular biology. Meanwhile, SBs are associated with various metabolic diseases such as Type 2 Diabetes mellitus (T2DM). Therefore, simultaneous quantitation of multiple SBs in biological samples could provide crucial information for uncovering underlying mechanisms of SBs related functions and diseases. However, existing methods are difficult to achieve simultaneous quantitation for multiple SBs due to the lack of isotope internal standards (ISs) of corresponding SBs. In the current study, we developed a highly sensitive method for the simultaneous detection of 26 SBs in biological samples by stable isotope labeling coupled with ultra-high performance liquid chromatography tandem mass spectrometry (SIL-UHPLC-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, 3-(N, N-dimethylamino)propyl isothiocyanate (DMPI) and d4-3-(N, N-dimethylamino)propyl isothiocyanate (d4-DMPI), were synthesized and utilized to label SBs in biological samples and SB standards, respectively. The d4-DMPI labeled SB standards were used as ISs to calibrate quantitation deviation in MS analysis from the biological matrix. Using the developed method, we successfully quantitated 19 SBs in cells, 20 SBs in mice feces and 18 SBs in human serum samples. Three C17-SBs used as ISs in many reported works were even found in all prepared samples. In summary, the developed SIL-UHPLC-MS/MS analysis was demonstrated to be a promising method for the simultaneous determination of multiple SBs, which could facilitate the investigation of cellular function of SBs and pathogenesis of related diseases.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Esfingolipídeos/sangue , Espectrometria de Massas em Tandem/métodos , Doença de Alzheimer/diagnóstico , Animais , Biomarcadores/sangue , Biomarcadores/química , Linhagem Celular Tumoral , Deutério , Diabetes Mellitus Tipo 2/diagnóstico , Fezes/química , Humanos , Isotiocianatos/química , Marcação por Isótopo/métodos , Limite de Detecção , Camundongos , Esfingolipídeos/química
9.
Nat Protoc ; 14(10): 2856-2877, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31471597

RESUMO

Precise quantification of metabolic pathway fluxes in biological systems is of major importance in guiding efforts in metabolic engineering, biotechnology, microbiology, human health, and cell culture. 13C metabolic flux analysis (13C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for 13C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with 13C-labeled glucose tracers, followed by gas chromatography-mass spectrometry (GC-MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for 13C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of fluxes. The presented protocol can be completed in 4 d and quantifies metabolic fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms.


Assuntos
Isótopos de Carbono/análise , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Marcação por Isótopo/métodos , Metabolômica/métodos , Isótopos de Carbono/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Glucose/metabolismo , Modelos Biológicos , Software , Fluxo de Trabalho
10.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1126-1127: 121738, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31377566

RESUMO

Myocardial Infarction (MI) is one of the most common causes of deaths worldwide. Thiols have been reported to play a key role in physiological and pathological processes of MI. Comprehensive analysis of thiols would be conducive to fully elucidate the relation between thiols and MI. In the current study, we analyze the metabolomic differences of thiols in serum between MI patients (n = 30) and healthy controls (HCs, n = 30) by stable isotope labeling-dispersive solid phase extraction-liquid chromatography-full scan-Orbitrap-mass spectrometry analysis (IL-DSPE-LC-full scan-Orbitrap MS) method. We detected 300 potential thiols in serum of MI patients and HCs, among which, 67 thiols were positively or putatively identified. Furthermore, we found that the levels of 71 thiols in serum exhibited significant difference between MI patients and HCs. In the transsulfuration pathway, we observed that Cys and Hcys were upregulated, while GSH were downregulated. Our results provide a comprehensive understanding of thiols metabolome in human serum between MI patients and HCs.


Assuntos
Metabolômica/métodos , Infarto do Miocárdio/metabolismo , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/metabolismo , Cromatografia Líquida/métodos , Análise por Conglomerados , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas , Metaboloma/fisiologia , Infarto do Miocárdio/sangue
11.
Anal Bioanal Chem ; 411(26): 6847-6856, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31440782

RESUMO

During drug development, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is used for visually elucidating the distribution of substances such as biomarkers, candidate compounds, and metabolites in the tissues. However, it is difficult to make relative comparisons between tissue sections and there are still many challenges. Here, we report a new method of "triple spray" for the comparison of analyte distribution in multiple tissue slices. This method targets amino acids and amines, and it incorporates the application of the internal standard in the on-tissue derivatization step. With further development, it has the potential to alleviate problems caused by the matrix effect. Initially, we measured three serial sections of rat brain to verify the efficacy of this method. In the hypothalamus, where gamma-aminobutyric acid (GABA) is known to be present in high concentration, the GABA levels of the three serial section showed little variation (CV = 1.62%). Subsequently, we compared the GABA level in the brain between stroke-prone spontaneous hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats with three individuals each. It showed significant differences between these models at the pre-selected region of interest (p < 0.05). Our results show that the triple spray allows for relative comparison among multiple tissue slices with high reproducibility. Graphical abstract.


Assuntos
Aminoácidos/análise , Química Encefálica , Neurotransmissores/análise , Animais , Indicadores e Reagentes , Marcação por Isótopo/métodos , Masculino , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ácido gama-Aminobutírico/análise
12.
Appl Microbiol Biotechnol ; 103(19): 8127-8143, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31420692

RESUMO

Chinese hamster ovary (CHO) cells are commonly used for the production of monoclonal antibodies. Omics technologies have been used to elucidate cellular switch points which result in higher monoclonal antibody (mAb) productivity and process yields in CHO and other biopharmaceutical production cell lines such as human or mouse. Currently, investigations of the phosphoproteome in CHO cell lines are rare yet could provide further insights into cellular mechanisms related to target product expression. Therefore, we investigated CHO IGF-signaling events using a comparative expression and phosphoproteomic approach in recombinant mAb-producing XL99 cell lines and corresponding parental strain. Differences were found on the level of protein expression between producer and parental cells in the exponential growth phase, mainly in proteins related to the lysosome, oligosaccharide metabolic processes, stress response, and cellular homeostasis. Within a stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic investigation of IGF signaling, expected general regulation of phosphorylation sites and cell line-specific responses were observed. Detected early phosphorylation events can be associated to observed effects of IGF on cellular growth, metabolism, and cell cycle distribution. Producer cell line-specific signaling exhibited differences to parental cells in intracellular trafficking and transcriptional processes, along with an overall lower amount of observable cross talk to other signaling pathways. By combining label-free and SILAC-based expression for phosphoproteomic analyses, cellular differences in the highly interactive levels of signaling and protein expression were detected, indicating alterations in metabolism and growth following treatment with an exogenous growth factor. The characterization of cell lines and effects of IGF addition resulted in identification of metabolic switch points. With this data, it will be possible to modulate pathways towards increased CHO process yield by targeted application of small-molecule inhibitors.


Assuntos
Células CHO/metabolismo , Marcação por Isótopo/métodos , Fosfoproteínas/análise , Proteoma/análise , Proteômica/métodos , Transdução de Sinais , Somatomedinas/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Cricetulus , Espectrometria de Massas/métodos , Proteínas Recombinantes/biossíntese
13.
Nat Commun ; 10(1): 2584, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197144

RESUMO

The Type VI secretion system (T6SS) is important for bacterial competition as well as virulence in many Gram-negative bacteria and its dynamics and regulation varies significantly between species. To gain insights into the mechanisms regulating T6SS assembly, we apply targeted proteomics to determine the abundance of the key T6SS components in Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. We show that while there are species specific exceptions, the abundance of most components is similar in all three bacteria and ranges from less than hundred to tens of thousands of copies per cell. The comparison of T6SS dynamics and protein abundance in V. cholerae grown under various conditions suggests that the critical component TssE and the secreted protein VasX are unstable and this diminishes T6SS assembly when protein synthesis is limited. Our quantitative analysis opens possibilities to build realistic models of T6SS assembly and to identify principles of T6SS regulation in various species.


Assuntos
Proteínas de Bactérias/análise , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo VI/análise , Proteínas de Bactérias/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Estabilidade Proteica , Proteômica/métodos , Sistemas de Secreção Tipo VI/metabolismo
14.
Nat Protoc ; 14(7): 1970-1990, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168088

RESUMO

Identification of previously unreported metabolites (so-called 'unknowns') in untargeted metabolomic data has become an increasingly active area of research. Considerably less attention, however, has been dedicated to identifying unknown metabolic pathways. Yet, for each unknown metabolite structure, there is potentially a yet-to-be-discovered chemical transformation. Elucidating these biochemical connections is essential to advancing our knowledge of cellular metabolism and can be achieved by tracking an isotopically labeled precursor to an unexpected product. In addition to their role in mapping metabolic fates, isotopic labels also provide critical insight into pathway dynamics (i.e., metabolic fluxes) that cannot be obtained from conventional label-free metabolomic analyses. When labeling is compared quantitatively between conditions, for example, isotopic tracers can enable relative pathway activities to be inferred. To discover unexpected chemical transformations or unanticipated differences in metabolic pathway activities, we have developed X13CMS, a platform for analyzing liquid chromatography/mass spectrometry (LC/MS) data at the systems level. After providing cells, animals, or patients with an isotopically enriched metabolite (e.g., 13C, 15N, or 2H), X13CMS identifies compounds that have incorporated the isotopic tracer and reports the extent of labeling for each. The analysis can be performed with a single condition, or isotopic fates can be compared between multiple conditions. The choice of which metabolite to enrich and which isotopic label to use is highly context dependent, but 13C-glucose and 13C-glutamine are often applied because they feed a large number of metabolic pathways. X13CMS is freely available.


Assuntos
Marcação por Isótopo/métodos , Metabolômica/métodos , Software , Animais , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Cromatografia Líquida/métodos , Neoplasias Colorretais/metabolismo , Fibroblastos/metabolismo , Humanos , Ácido Láctico/metabolismo , Espectrometria de Massas/métodos , Palmitatos/metabolismo , Fluxo de Trabalho
15.
Eur J Pharm Biopharm ; 140: 109-120, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082509

RESUMO

Radionuclide molecular imaging is a promising tool that becomes increasingly important as targeted cancer therapies are developed. To ensure an effective treatment, a molecular stratification of the cancer is a necessity. To accomplish this, visualization of cancer associated molecular abnormalities in vivo by molecular imaging is the method of choice. ADAPTs, a novel type of small protein scaffold, have been utilized to select and develop high affinity binders to different proteinaceous targets. One of these binders, ADAPT6 selectively interacts with human epidermal growth factor 2 (HER2) with low nanomolar affinity and can therefore be used for its in vivo visualization. Molecular design and optimization of labeled anti-HER2 ADAPT has been explored in several earlier studies, showing that small changes in the scaffold affect the biodistribution of the domain. In this study, we evaluate how the biodistribution properties of ADAPT6 is affected by the commonly used maleimido derivatives of the macrocyclic chelators NOTA, NODAGA, DOTA and DOTAGA with the aim to select the best variants for SPECT and PET imaging. The different conjugates were labeled with 111In for SPECT and 68Ga for PET. The acquired data show that the combination of a radionuclide and a chelator for its conjugation has a strong influence on the uptake of ADAPT6 in normal tissues and thereby gives a significant variation in tumor-to-organ ratios. Hence, it was concluded that the best variant for SPECT imaging is 111In-(HE)3DANS-ADAPT6-GSSC-DOTA while the best variant for PET imaging is 68Ga-(HE)3DANS-ADAPT6-GSSC-NODAGA.


Assuntos
Quelantes/química , Radioisótopos de Gálio/química , Radioisótopos de Índio/química , Compostos Macrocíclicos/química , Proteínas/metabolismo , Cintilografia/métodos , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo/métodos , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Distribuição Tecidual/efeitos dos fármacos , Tomografia Computadorizada de Emissão de Fóton Único/métodos
16.
Methods Mol Biol ; 1978: 199-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119665

RESUMO

Arginine metabolism is linked to several important metabolic processes, and reprogramming of arginine metabolism occurs in various physiological and pathological conditions. Here we describe a method, using a LC-MS-based metabolomics and 15N4-arginine tracing approach, to quantitatively analyze arginine metabolism. This method can reliably quantify the abundance of important intermediates and fluxes of major metabolic reactions in arginine metabolism in a variety of cultured mammalian cell models.


Assuntos
Cromatografia Líquida/métodos , Marcação por Isótopo/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Arginina/metabolismo , Isótopos de Carbono/química , Humanos
17.
Methods Mol Biol ; 1978: 219-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119666

RESUMO

Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply 13C isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.


Assuntos
Técnicas de Cultura de Células/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Marcação por Isótopo/métodos , Lipogênese/genética , Animais , Isótopos de Carbono/química , Glutamina/metabolismo , Humanos , Redes e Vias Metabólicas/genética
18.
Methods Mol Biol ; 1978: 269-283, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119669

RESUMO

Stable isotope tracing allows a metabolic substrate to be followed through downstream biochemical reactions, thereby providing unparalleled insights into the metabolic wiring of cells. This approach stops short of modeling absolute fluxes but is relatively straightforward and has become increasingly accessible due to the widespread adoption of high-resolution mass spectrometers. Analysis of both dynamic and steady-state labeling patterns in downstream metabolites provides valuable qualitative information as to their origin and relative rates of production. Stable isotope tracing is, therefore, a powerful way to understand the impact of genetic alterations and defined perturbations on metabolism. In this chapter, we describe a liquid chromatography-mass spectrometry (LC-MS) protocol for stable isotope tracing using 13C-L-arginine in a macrophage cell line. A similar approach can be used to follow other stable isotope tracers, and notes are provided with advice on how this protocol can be generalized for use in other settings.


Assuntos
Cromatografia Líquida/métodos , Marcação por Isótopo/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/química , Redes e Vias Metabólicas/genética
19.
Methods Enzymol ; 620: 145-166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31072485

RESUMO

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed.


Assuntos
Ensaios Enzimáticos/métodos , Marcação por Isótopo/métodos , Oxirredutases/química , Dicroísmo Circular , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Cinética , Isótopos de Nitrogênio/química , Oxirredutases/genética , Oxirredutases/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
J Am Soc Mass Spectrom ; 30(9): 1733-1741, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31140076

RESUMO

Chemical isotope labeling (CIL) LC-MS is a highly sensitive and quantitative method for metabolome analysis. Because of a large number of peaks detectable in a sample and the need of running many samples in a metabolomics project, any significant change in mass measurement accuracy during the whole period of running samples can adversely affect the downstream peak alignment and quantitative analysis. Herein, we report a rapid method to check the mass accuracy of individual spectra in each CIL LC-MS run in order to flag up any run containing spectra with accuracy drift that falls outside the expected error. The flagged run may be re-run or discarded before merging with other runs for peak alignment and analysis. This method is based on the observation that some background signals are commonly detected in almost all spectra collected in CIL LC-MS runs. A mass accuracy check (MAC) software program has been developed to first find the common background mass peaks and then use them as mass references to calculate any mass shifts over the course of multiple sample runs. Using a metabolome dataset of 324 human cerebrospinal fluid (CSF) samples and 35 quality control (QC) samples produced by CIL LC-MS, we show that this accuracy check method can streamline the initial raw data processing for downstream analysis in metabolomics.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodos , Software , Calibragem , Isótopos de Carbono/química , Líquido Cefalorraquidiano/metabolismo , Compostos de Dansil/química , Confiabilidade dos Dados , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas/normas , Metabolômica/normas , Processamento de Sinais Assistido por Computador , Traumatismos da Medula Espinal/líquido cefalorraquidiano
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA