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1.
J Chromatogr A ; 1626: 461324, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797817

RESUMO

Sorption of PFASs onto surfaces of laboratory materials has been frequently reported. Due to the often complex and poorly understood nature of such sorption, workarounds have often included use of whole samples only, accompanied by sample vessel rinsing to desorb active surfaces. The resulting methods tend to require considerable sample preparation times and preclude typical activities such as aliquoting and dilution of water samples prior to extraction. This manuscript reports an approach for PFAS analysis which uses subsampling of water matrices from vessels including centrifuge tubes and autosampler vials, through the optimized use of solvent to reduce PFAS retention on subsampling vessels. Online solid phase extraction (SPE) using a weak anion exchange resin is then used to concentrate sample aliquots to improve sensitivity and allow for removal of matrix interferences. With the technique of ultra performance liquid chromatography (UPLC) coupled to isotope dilution tandem mass spectrometry, statistically based quantitation limits ranged from sub ng/L to single digit ng/L for carboxylate, sulfonate, and sulfonamide PFASs analytes from C4 to C12. Linear calibration ranges were from 0.25 to 4000 ng/L. Matrix effects relevant for drinking water treatment studies, such as cations, organic carbon, and competing PFAS compounds, were evaluated and found to not impact method performance within QC criteria consistent with study data quality objectives.


Assuntos
Fluorcarbonetos/análise , Indicadores e Reagentes/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão , Fluorcarbonetos/isolamento & purificação , Água Doce/análise , Indicadores e Reagentes/isolamento & purificação , Marcação por Isótopo , Sais/química , Extração em Fase Sólida , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificação
2.
PLoS One ; 15(8): e0237612, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790731

RESUMO

Seventeen glass vessels and twenty glass beads recovered from the excavations at the ancient city of Malindi and the archaeological site of Mambrui in Kenya, east Africa were analysed using electron probe microanalysis (EPMA) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The results show that all of the glass samples are soda-lime-silica glass. They belong to the high alumina -plant ash glass type, characterised by high alumina and relatively low calcium contents, widely distributed in eastern (10th- 16th centuries AD) and southern Africa (13th - 15th centuries AD), Central Asia (9th- 14th centuries AD) and southeast Asia (12th- 13th centuries AD), made with plant ashes and sands. This is an understudied glass type for which previous research has indicated there were three types. When compared with published research on such glasses using Zr, Ti, Ba, Cr, La, Li, Cs, Na2O, MgO and CaO we have identified at least four different compositional groups of v-Na-Al glass: Types A, B, C and D. By comparing the results with contemporary v-Na-Al glass vessels and beads from Central Asia, Africa, and southeast Asia we show that most of the Malindi and Mambrui glass share similar characteristics to the compositions of Mapungubwe Oblate and some of the Madagascar glass beads from southern Africa. They belong to Type A v-Na-Al glass which is characterised by an elevated level of Ti and Ba and a relatively high ratios of Cr/La, relatively low Zr concentrations and low ratios of Zr/Ti. Differences in Zr, Li, MgO and Na2O concentrations in Type A glass indicates that there are subgroups which might derive from different glass workshop(s) specialising in Type A v-Na-Al glass production. Comparison with the chemical compositions of glass from Ghazni, Afghanistan and Termez, Uzbekistan, and by using lead isotope analysis, we suggest v-Na-Al glass was manufactured in Central Asia and possibly worked into vessels and beads there.


Assuntos
Compostos de Cálcio/química , Vidro/química , Óxidos/química , Plantas/química , Dióxido de Silício/química , Hidróxido de Sódio/química , Oligoelementos/análise , Oligoelementos/história , África Oriental , Óxido de Alumínio , Arqueologia , História do Século XV , História do Século XVI , Oceano Índico , Marcação por Isótopo/métodos , Quênia , Espectrometria de Massas
3.
J Chromatogr A ; 1626: 461333, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797819

RESUMO

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.


Assuntos
Nicotina/sangue , Anabasina/sangue , Anabasina/isolamento & purificação , Anabasina/normas , Animais , Cromatografia Líquida de Alta Pressão/normas , Cotinina/análogos & derivados , Cotinina/sangue , Cotinina/isolamento & purificação , Cotinina/normas , Marcação por Isótopo , Limite de Detecção , Nicotina/análogos & derivados , Nicotina/isolamento & purificação , Nicotina/metabolismo , Nicotina/normas , Coelhos , Padrões de Referência , Reprodutibilidade dos Testes , Abandono do Hábito de Fumar , Microextração em Fase Sólida , Espectrometria de Massas em Tandem/normas , Fatores de Tempo
4.
PLoS One ; 15(8): e0237850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853240

RESUMO

Individual geographic mobility is a key social dynamic of early Viking-Age urbanization in Scandinavia. We present the first comprehensive geographic mobility study of Scandinavia's earliest emporium, Ribe, which emerged around AD 700 in the North Sea region of Denmark. This article presents the results of strontium isotope analyses of 21 individuals buried at Ribe, combined with an in-depth study of the varied cultural affinities reflected by the burial practices. In order to investigate geographic mobility in early life/childhood, we sampled multiple teeth and/or petrous bone of individuals, which yielded a total of 43 strontium isotope analyses. Most individuals yielded strontium isotope values that fell within a relatively narrow range, between 87Sr/86Sr = 0.709 to 0.711. Only two individuals yielded values >87Sr/86Sr = 0.711. This suggests that most of these individuals had local origins but some had cultural affinities beyond present-day Denmark. Our results raise new questions concerning our understanding of the social and cultural dynamics behind the urbanization of Scandinavia.


Assuntos
Sepultamento , Geografia , Marcação por Isótopo , Isótopos de Estrôncio/análise , Alemanha , Humanos , Países Escandinavos e Nórdicos
5.
BMC Bioinformatics ; 21(1): 297, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650717

RESUMO

BACKGROUND: Stable isotope tracing has become an invaluable tool for probing the metabolism of biological systems. However, data analysis and visualization from metabolic tracing studies often involve multiple software packages and lack pathway architecture. A deep understanding of the metabolic contexts from such datasets is required for biological interpretation. Currently, there is no single software package that allows researchers to analyze and integrate stable isotope tracing data into annotated or custom-built metabolic networks. RESULTS: We built a standalone web-based software, Escher-Trace, for analyzing tracing data and communicating results. Escher-Trace allows users to upload baseline corrected mass spectrometer (MS) tracing data and correct for natural isotope abundance, generate publication quality graphs of metabolite labeling, and present data in the context of annotated metabolic pathways. Here we provide a detailed walk-through of how to incorporate and visualize 13C metabolic tracing data into the Escher-Trace platform. CONCLUSIONS: Escher-Trace is an open-source software for analysis and interpretation of stable isotope tracing data and is available at https://escher-trace.github.io/ .


Assuntos
Marcação por Isótopo/métodos , Redes e Vias Metabólicas , Software , Gráficos por Computador , Espectrometria de Massas/métodos
6.
PLoS One ; 15(7): e0235752, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667954

RESUMO

We have limited knowledge of the patterns, causes, and prevalence of elevational migration despite observations of seasonal movements of animals along elevational gradients in montane systems worldwide. While a third of extant Hawaiian landbird species are estimated to be elevational migrants this assumption is based primarily on early naturalist's observations with limited empirical evidence. In this study, we compared stable hydrogen isotopes (δ2H) of metabolically inert (feathers) and active (blood plasma, red blood cells) tissues collected from the same individual to determine if present day populations of Hawaiian honeycreepers undergo elevational movements to track areas of seasonally high flower bloom that constitute significant food resources. We also measured stable carbon isotopes (δ13C) and stable nitrogen isotopes (δ15N) to examine potential changes in diet between time periods. We found that the majority of 'apapane (Himatione sanguinea) and Hawai'i 'amakihi (Chlorodrepanis virens) captured at high elevation, high bloom flowering sites in the fall were not year-round residents at the capture locations, but had molted their feathers at lower elevations presumably in the summer after breeding. δ2H values of feathers for all individuals sampled were higher than blood plasma isotope values after accounting for differences in tissue-specific discrimination. We did not find a difference in the propensity of elevational movement between 'apapane and Hawai'i 'amakihi, even though the 'amakihi is considered more sedentary. However, consistent with a more generalist diet, δ15N values indicated that Hawai'i 'amakihi had a more diverse diet across trophic levels than 'apapane, and a greater reliance on nectar in the fall. We demonstrate that collecting multiple tissue samples, which grow at different rates or time periods, from a single individual can provide insights into elevational movements of Hawaiian honeycreepers over an extended time period.


Assuntos
Migração Animal/fisiologia , Radioisótopos de Carbono/análise , Eritrócitos/metabolismo , Plumas/metabolismo , Marcação por Isótopo/métodos , Radioisótopos de Nitrogênio/análise , Passeriformes/fisiologia , Animais , Hawaii , Dinâmica Populacional
7.
Food Chem ; 332: 127413, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32652410

RESUMO

Mass spectrometry-based methods coupled with stable isotope dilution have become effective and widely used methods for the detection and quantification of food allergens. Current methods target signature peptides resulting from proteolytic digestion of proteins of the allergenic ingredient. The choice of appropriate stable isotope-labelled internal standard is crucial, given the diversity of encountered food matrices which can affect sample preparation and analysis. We propose the use of concatemer, an artificial and stable isotope-labelled protein composed of several concatenated signature peptides as internal standard. With a comparative analysis of three matrices contaminated with four allergens (egg, milk, peanut, and hazelnut), the concatemer approach was found to offer advantages associated with the use of labelled proteins, ideal but unaffordable, and circumvent certain limitations of traditionally used synthetic peptides as internal standards. Although used in the proteomic field for more than a decade, concatemer strategy has not yet been applied for food analysis.


Assuntos
Alérgenos/análise , Proteômica/métodos , Alérgenos/metabolismo , Sequência de Aminoácidos , Animais , Arachis/metabolismo , Cromatografia Líquida de Alta Pressão , Corylus/metabolismo , Ovos/análise , Marcação por Isótopo , Espectrometria de Massas , Leite/metabolismo , Isótopos de Nitrogênio/química , Peptídeos/análise , Peptídeos/química , Proteômica/normas , Padrões de Referência , Espectrometria de Massas em Tandem
8.
Proc Natl Acad Sci U S A ; 117(30): 17710-17719, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661161

RESUMO

Cat remains from Poland dated to 4,200 to 2,300 y BCE are currently the earliest evidence for the migration of the Near Eastern cat (NE cat), the ancestor of domestic cats, into Central Europe. This early immigration preceded the known establishment of housecat populations in the region by around 3,000 y. One hypothesis assumed that NE cats followed the migration of early farmers as synanthropes. In this study, we analyze the stable isotopes in six samples of Late Neolithic NE cat bones and further 34 of the associated fauna, including the European wildcat. We approximate the diet and trophic ecology of Late Neolithic felids in a broad context of contemporary wild and domestic animals and humans. In addition, we compared the ecology of Late Neolithic NE cats with the earliest domestic cats known from the territory of Poland, dating to the Roman Period. Our results reveal that human agricultural activity during the Late Neolithic had already impacted the isotopic signature of rodents in the ecosystem. These synanthropic pests constituted a significant proportion of the NE cat's diet. Our interpretation is that Late Neolithic NE cats were opportunistic synanthropes, most probably free-living individuals (i.e., not directly relying on a human food supply). We explore niche partitioning between studied NE cats and the contemporary native European wildcats. We find only minor differences between the isotopic ecology of both these taxa. We conclude that, after the appearance of the NE cat, both felid taxa shared the ecological niches.


Assuntos
Animais Domésticos , Arqueologia , Dieta , Fósseis , Animais , Gatos , Colágeno , Ecossistema , Europa (Continente) , Geografia , Humanos , Marcação por Isótopo
9.
Am J Clin Nutr ; 112(3): 576-585, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32614379

RESUMO

BACKGROUND: Maternal iron absorption during pregnancy can be evaluated using RBC incorporation of orally administered stable iron isotope. This approach underestimates true maternal absorption of iron as it does not account for absorbed iron that is transferred to the fetus or retained within the placenta. OBJECTIVE: Our objective was to re-evaluate maternal iron absorption after factoring in these losses and identify factors associated with iron partitioning between the maternal, neonatal, and placental compartments. METHODS: This study utilized data from stable iron isotope studies carried out in 68 women during the third trimester of pregnancy. Iron status indicators and stable iron isotopic enrichment were measured in maternal blood, umbilical cord blood, and placental tissue when available. Factors associated with iron isotope partitioning between the maternal, neonatal, and placental compartments were identified. RESULTS: On average, true maternal absorption of iron increased by 10% (from 19% to 21%) after accounting for absorbed iron present in the newborn (P < 0.001), and further increased by 7%, (from 39% to 42%, P < 0.001) after accounting for iron retained within the placenta. On average, 2% of recovered tracer was present in the placenta and 6% was found in the newborn. Net transfer of iron to the neonate was higher in women with lower total body iron (standardized ß = -0.48, P < 0.01) and lower maternal hepcidin (standardized ß = -0.66, P < 0.01). In women carrying multiple fetuses, neonatal hepcidin explained a significant amount of observed variance in net placental transfer of absorbed iron (R = 0.95, P = 0.03). CONCLUSIONS: Maternal RBC iron incorporation of an orally ingested tracer underestimated true maternal iron absorption. The degree of underestimation was greatest in women with low body iron. Maternal hepcidin was inversely associated with maternal RBC iron utilization, whereas neonatal hepcidin explained variance in net transfer of iron to the neonatal compartment.These trials were registered at clinicaltrials.gov as NCT01019096 and NCT01582802.


Assuntos
Feto/metabolismo , Ferro/farmacocinética , Placenta/metabolismo , Adolescente , Adulto , Transporte Biológico , Feminino , Humanos , Recém-Nascido , Ferro/metabolismo , Isótopos de Ferro , Marcação por Isótopo , Gravidez , Adulto Jovem
10.
J Chromatogr A ; 1623: 461182, 2020 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-32505283

RESUMO

Hydroxyl-polycyclic aromatic hydrocarbons (OH-PAHs) are biomarkers for assessing the exposure levels of polycyclic aromatic hydrocarbons (PAHs). A series of stable isotope mass tags (SIMT-332/338/346/349/351/354/360/363/374/377) were firstly designed and synthesized to perform multiplexed stable isotope labeling derivatization (MSILD) of OH-PAHs in human plasma and urine. Their derivatives were enriched and purified by magnetic dispersive solid phase extraction (MDSPE) using prepared Fe3O4/GO and then determined by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in multiple reaction monitoring mode. 9-Plexed MSILD reagents were prepared using pipemidic acid as core structure with different isotope mass tags, in which carbonyl chloride group was used to label OH-PAHs. The SIMT-346 labeled OH-PAHs standards were used as internal standards, which can greatly increase the quantitative accuracy. 9-Plex labeled nine different real samples can be quantified by UHPLC-MS/MS in a single run. Under optimized MSILD-MDSPE conditions, good linearities of seven OH-PAHs were obtained with satisfactory coefficient of determination R2 > 0.991. Limits of detection (LODs) of seven OH-PAHs were from 0.1 to 0.5 pg/mL, and limits of quantitation (LOQs) ranged from 0.5 to 2.0 pg/mL. The intra- and inter-day precisions ranged in 2.3-12.4% with accuracies in the range of 91.7-108.4%. Acceptable results of matrix effect (89.7-105.7%) and derivatization efficiency (> 96.4%) were obtained. In short, the developed method has been proved to be high-throughput, sensitive, accurate and easy-handling. This method was applied for the measurement of seven free OH-PAHs in human urine and plasma, and expected to provide technical support for the evaluation of PAHs exposure levels in humans.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hidrocarbonetos Policíclicos Aromáticos/sangue , Hidrocarbonetos Policíclicos Aromáticos/urina , Espectrometria de Massas em Tandem/métodos , Adsorção , Humanos , Concentração de Íons de Hidrogênio , Marcação por Isótopo , Limite de Detecção , Hidrocarbonetos Policíclicos Aromáticos/química , Reprodutibilidade dos Testes
11.
Ann Biol Clin (Paris) ; 78(3): 319-322, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32540818

RESUMO

The purpose of this work was to compare the measured red-cell volume (RCV) using sodium pertechnétate [RCV-99mTc] compared to the reference technique using sodium radiochromate [RCV-51Cr] and to assess the influence of technetium-99 elution on the RCV-99mTc value. Ten patients had simultaneous measurements of RCV-99mTc and RCV-51Cr. Elution of Tc-99m from red blood cells was 2.9% and led to an average overestimation of RCV-99mTc of 3.7%. The introduction of individual tracer elution rates in the RCV-99mTc calculation corrects this overestimation.


Assuntos
Radioisótopos de Cromo/farmacologia , Volume de Eritrócitos/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Marcação por Isótopo/métodos , Tecnécio/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Contagem de Eritrócitos/métodos , Feminino , Hematócrito/métodos , Humanos , Marcação por Isótopo/efeitos adversos , Masculino , Pessoa de Meia-Idade , Técnica de Diluição de Radioisótopos
12.
PLoS One ; 15(6): e0235114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574188

RESUMO

Nursing and weaning periods are poorly understood in cetaceans due to the difficulty of assessing underwater behaviour in the wild. However, the onset and completion of weaning are critical turning points for individual development and survival, with implications for a species' life history including reproductive potential. δ15N and δ13C deposited in odontocete teeth annuli provide a lifetime record of diet, offering an opportunity to investigate variation and trends in fundamental biology. While available reproductive parameters for beaked whales have largely been inferred from single records of stranded or hunted animals and extrapolated across species, here we examine the weaning strategy and nursing duration in northern bottlenose whales (Hyperoodon ampullatus) by measuring stable isotopes deposited in dentine growth layer groups (GLGs). Using a collection of H. ampullatus teeth taken from whales killed during the whaling era (N = 48) and from two stranded specimens, we compared ontogenetic variation of δ15N and δ13C found in annual GLGs across all individuals, by sex and by region. We detected age-based trends in both δ15N and δ13C that are consistent across regions and males and females, and indicate that nursing is prolonged and weaning does not conclude until whales are 3-4 years old, substantially later than previous estimates of 1 year. Incorporating a prolonged period of maternal care into H. ampullatus life history significantly reduces their reproductive potential, with broad implications for models of beaked whale life history, energetics and the species' recovery from whaling.


Assuntos
Dentina/metabolismo , Marcação por Isótopo/métodos , Reprodução/fisiologia , Baleias/fisiologia , Animais , Animais Recém-Nascidos , Isótopos de Carbono/metabolismo , Feminino , Geografia , Islândia , Masculino , Terra Nova e Labrador , Isótopos de Nitrogênio/metabolismo , História Reprodutiva
13.
Curr Opin Plant Biol ; 55: 84-92, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32388402

RESUMO

The exact mechanics of specialized metabolism and its importance throughout plant evolution remain mysterious. Specialized metabolites and their corresponding biosynthetic genes are crucial to understand the reason for the prevalence of certain metabolism. Even though mass spectrometry-based metabolomics has enabled us to acquire data about the structural properties of unknown specialized metabolites as well as known metabolites and their corresponding isomers/analogs, extensive analytical approaches are still required. Herein, we review the most advanced analytical approaches using stable isotope labeling that can be used to identify the unknown specialized metabolites.


Assuntos
Metabolômica , Plantas , Marcação por Isótopo , Espectrometria de Massas
14.
J Vis Exp ; (159)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32449746

RESUMO

Many nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N∙ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.


Assuntos
Marcação por Isótopo/métodos , Isótopos de Nitrogênio/análise , Solo/química , Zea mays/química , Biomassa , Fertilizantes/análise
15.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 326-331, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254056

RESUMO

The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallography has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid-state nuclear magnetic resonance and two-dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular-dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.


Assuntos
Simulação de Dinâmica Molecular , Canais de Potássio/química , Potássio/química , Cristalografia por Raios X , Marcação por Isótopo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Espalhamento de Radiação , Espectrofotometria Infravermelho
16.
Inorg Chem ; 59(8): 5728-5741, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32242663

RESUMO

[44/47Sc]Sc3+, [68Ga]Ga3+, and [111In]In3+ are the three most attractive trivalent smaller radiometalnuclides, offering a wide range of distinct properties (emission energies and types) in the toolbox of nuclear medicine. In this study, all three of the metal ions are successfully chelated using a new oxine-based hexadentate ligand, H3glyox, which forms thermodynamically stable neutral complexes with exceptionally high pM values [pIn (34) > pSc (26) > pGa (24.9)]. X-ray diffraction single crystal structures with stable isotopes revealed that the ligand is highly preorganized and has a perfect fit to size cavity to form [Sc(glyox)(H2O)] and [In(glyox)(H2O)] complexes. Quantitative radiolabeling with gallium-68 (RCY > 95%, [L] = 10-5 M) and indium-111 (RCY > 99%, [L] = 10-8 M) was achieved under ambient conditions (RT, pH 7, and 15 min) with very high apparent molar activities of 750 MBq/µmol and 650 MBq/nmol, respectively. Preliminary quantitative radiolabeling of [44Sc]ScCl3 (RCY > 99%, [L] = 10-6 M) was fast at room temperature (pH 7 and 10 min). In vitro experiments revealed exceptional stability of both [68Ga]Ga(glyox) and [111In]In(glyox) complexes against human serum (transchelation <2%) and its suitability for biological applications. Additionally, on chelation with metal ions, H3glyox exhibits enhanced fluorescence, which was employed to determine the stability constants for Sc(glyox) in addition to the in-batch UV-vis spectrophotometric titrations; as a proof-of-concept these complexes were used to obtain fluorescence images of live HeLa cells using Sc(glyox) and Ga(glyox), confirming the viability of the cells. These initial investigations suggest H3glyox to be a valuable chelator for radiometal-based diagnosis (nuclear and optical imaging) and therapy.


Assuntos
Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Corantes Fluorescentes/farmacologia , Oximas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Quelantes/síntese química , Complexos de Coordenação/sangue , Complexos de Coordenação/química , Estabilidade de Medicamentos , Corantes Fluorescentes/química , Radioisótopos de Gálio/química , Células HeLa , Humanos , Radioisótopos de Índio/química , Marcação por Isótopo , Ligantes , Microscopia de Fluorescência/métodos , Oximas/síntese química , Estudo de Prova de Conceito , Radioisótopos/química , Compostos Radiofarmacêuticos/sangue , Compostos Radiofarmacêuticos/química , Escândio/química , Termodinâmica
17.
J Vis Exp ; (157)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32250355

RESUMO

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine ('K-ε-diglycine' or simply 'diGly') can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteínas Ubiquitinadas/análise , Ubiquitinação , Sequência de Aminoácidos , Animais , Bortezomib/farmacologia , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Camundongos , Peptídeos/química , Proteínas Ubiquitinadas/química
18.
Cell Mol Life Sci ; 77(17): 3293-3309, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32130428

RESUMO

Metallomics is a rapidly evolving field of bio-metal research that integrates techniques and perspectives from other "-omics" sciences (e.g. genomics, proteomics) and from research vocations further afield. Perhaps the most esoteric of this latter category has been the recent coupling of biomedicine with element and isotope geochemistry, commonly referred to as isotope metallomics. Over the course of less than two decades, isotope metallomics has produced numerous benchmark studies highlighting the use of stable metal isotope distribution in developing disease diagnostics-e.g. cancer, neurodegeneration, osteoporosis-as well as their utility in deciphering the underlying mechanisms of such diseases. These pioneering works indicate an enormous wealth of potential and provide a call to action for researchers to combine and leverage expertise and resources to create a clear and meaningful path forward. Doing so with efficacy and impact will require not only building on existing research, but also broadening collaborative networks, bolstering and deepening cross-disciplinary channels, and establishing unified and realizable objectives. The aim of this review is to briefly summarize the field and its underpinnings, provide a directory of the state of the art, outline the most encouraging paths forward, including their limitations, outlook and speculative upcoming breakthroughs, and finally to offer a vision of how to cultivate isotope metallomics for an impactful future.


Assuntos
Metais/metabolismo , Pesquisa Biomédica , Cromatografia por Troca Iônica , Humanos , Marcação por Isótopo , Espectrometria de Massas , Metais/análise , Neoplasias/metabolismo , Neoplasias/patologia , Osteoporose/metabolismo , Osteoporose/patologia
19.
Can J Microbiol ; 66(8): 491-494, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32134703

RESUMO

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.


Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Isótopos de Carbono/química , Centrifugação com Gradiente de Concentração/métodos , Escherichia coli/química , Formamidas/química , Marcação por Isótopo/métodos , RNA Bacteriano/química , Ultracentrifugação/métodos
20.
Nat Methods ; 17(4): 399-404, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203386

RESUMO

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.


Assuntos
Peptídeos/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Humanos , Marcação por Isótopo
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