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1.
Artigo em Inglês | MEDLINE | ID: mdl-31990641

RESUMO

Condensed phosphates are deliberately added to the fishery and processed marine food products on purpose to increase the weight of products. However, excessive intake overwhelming bodily homoeostasis can result in phosphate toxicity such as mineral and bone disorders, associated with chronic kidney diseases, and cardiovascular events. Rapid analysis for condensed phosphates is required to detect the illegal adulteration of processed marine products. We optimised an analytical method using ion chromatography for the rapid and selective detection of condensed phosphates in various kinds of fishery and processed marine products. We compared the performance of three columns (IonPac AS11, AS11-HC, and AS16) for the detection of condensed phosphates with respect to time of analysis and sensitivity. The IonPac AS11 column exhibited the shortest retention time for the major condensed phosphates (pyro-, tri-, and trimetaphosphate), the highest sensitivity for trimetaphosphate, and good repeatability and precision. Microwave and boiling processing were examined as methods to prevent hydrolysis of condensed phosphates into orthophosphate, which is critical in distinguishing intentionally added condensed phosphates from naturally occurring orthophosphate. Microwave treatment was determined to be the more effective method to suppress hydrolysis of condensed phosphates to orthophosphate. Furthermore, microwave processing could be used for thawing the frozen samples, saving extra effort and time. We confirmed that the method exhibits good recovery (80% or more) and precision (%RSD < 10%) for samples with various matrices. With the method, 14 kinds of fishery and processed marine food products were successfully analysed for condensed phosphates. Especially, we identified that phosphate levels in the processed shrimp and dried shredded squid samples exceeded the maximum allowable levels specified in the CODEX standard. We believe that our method would be useful for the rapid analysis of condensed phosphates in various types of fishery and processed marine food products.


Assuntos
Fosfatos/análise , Animais , Cromatografia por Troca Iônica , Pesqueiros , Hidrolases/metabolismo , Limite de Detecção , Micro-Ondas , Alimentos Marinhos , Sensibilidade e Especificidade , Extração em Fase Sólida
2.
J Chromatogr A ; 1609: 460429, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31431354

RESUMO

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column's full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone's action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone's travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3-6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5-fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2-9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two 'protein-free' systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the 'state-of-the-art' technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.


Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Proteína Estafilocócica A/análise , Temperatura Ambiente , Tampões (Química) , Cromatografia por Troca Iônica/métodos , Dicroísmo Circular , Condutividade Elétrica , Temperatura Alta , Estabilidade Proteica
3.
J Chromatogr Sci ; 57(10): 939-943, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31665235

RESUMO

Chromium exists in its two stable oxidation states including trivalent chromium (Cr (III)) and hexavalent chromium (Cr (VI)) in natural waters. Chromium is an essential micronutrient in the trivalent form; however, the hexavalent form of chromium is considered to be a carcinogen. It is important to determine the chromium content along with speciation. There are a number of methods available for chromium determination. Speciation of chromium is essential to know the exact composition of chromium. Ion exchange chromatography is one of the techniques used to determine Cr (VI). The proposed method can be used to perform the speciation of Cr (III) and Cr (VI). It is a two-step process: first Cr (VI) is determined, followed by total chromium determination by treating the sample with potassium permanganate solution to oxidize the Cr (III) present in the sample to Cr (VI) and determining it as Cr (VI). Conductivity detector is used for the detection. Addition of potassium permanganate solution to the ground water samples for oxidizing the Cr (III) to Cr (VI) is the newly adopted sample preparation technique. The effect of potassium permanganate oxidation is studied in detail in the proposed method. This method can be used for chromium speciation in river water and ground water samples.


Assuntos
Cromatografia por Troca Iônica/métodos , Cromo/análise , Cromo/química , Condutividade Elétrica , Limite de Detecção , Modelos Lineares , Oxirredução , Permanganato de Potássio/química , Reprodutibilidade dos Testes
4.
Anal Bioanal Chem ; 412(3): 681-690, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31834449

RESUMO

The development of mercury (Hg) stable isotope measurements has enhanced the study of Hg sources and transformations in the environment. As a result of the mixing of inorganic Hg (iHg) and methylmercury (MeHg) species within organisms of the aquatic food web, understanding species-specific Hg stable isotopic compositions is of significant importance. The lack of MeHg isotope measurements is due to the analytical difficulty in the separation of the MeHg from the total Hg pool, with only a few methods having been tested over the past decade with varying degrees of success, and only a handful of environmentally relevant measurements. Here, we present a novel anion-exchange resin separation method using AG 1-X4 that further isolates MeHg from the sample matrix, following a distillation pretreatment, in order to obtain ambient MeHg stable isotopic compositions. This method avoids the use of organic reagents, does not require complex instrumentation, and is applicable across matrices. Separation tests across sediment, water, and biotic matrices showed acceptable recoveries (98 ± 5%, n = 54) and reproducible δ202Hg isotope results (2 SDs ≤ 0.15‰) down to 5 ng of MeHg. The measured MeHg pools in natural matrices, such as plankton and sediments, showed large deviations from the non-speciated total Hg measurement, indicating that there is an important isotopic shift during methylation that is not recorded by typical measurements, but is vital in order to assess sources of Hg during bioaccumulation. Graphical abstract.


Assuntos
Cromatografia por Troca Iônica/métodos , Monitoramento Ambiental/métodos , Compostos de Metilmercúrio/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Animais , Peixes/metabolismo , Cadeia Alimentar , Sedimentos Geológicos/análise , Limite de Detecção , Isótopos de Mercúrio/análise , Isótopos de Mercúrio/isolamento & purificação , Compostos de Metilmercúrio/análise , Plâncton/química , Poluentes Químicos da Água/análise
5.
Food Chem ; 305: 125457, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505414

RESUMO

Taking into account a growing market and small number of articles related to honeydew honey, a metabolomic approach associated with multivariate analysis and modelling was proposed to discriminate five varieties of honey. Advanced analytical techniques were used for determination of 20 elements, 14 carbohydrates and stable carbon isotope ratio. No chemical marker has been found within sugar compounds, but several elements (Ba, Ca, Mg, Sr, Mn, Al, Co, Ni, Se) were marked as characteristic of honey type and allow classification of three botanical origins (Abies alba, Quercus frainetto, Quercus ilex). Sugars turanose, trehalose, arabinose and raffinose, elements Ba, Sr, P, Cd and Se, and δ13C values of honey, have different concentrations in honeys of the same botanical origin but harvested in different season. In addition to a confirmation of authenticity in terms of production, the values of δ13C of protein could be a good indicator of botanical origin.


Assuntos
Mel/análise , Espectrometria de Massas/métodos , Quercus/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Análise Discriminante , Análise dos Mínimos Quadrados , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Análise de Componente Principal , Álcoois Açúcares/análise , Açúcares/análise
6.
Artigo em Inglês | MEDLINE | ID: mdl-31877428

RESUMO

Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lycium/química , Espermidina , Espectrometria de Massas em Tandem/métodos , Alcaloides/análise , Alcaloides/química , Cromatografia por Troca Iônica/métodos , Extratos Vegetais/química , Extração em Fase Sólida/métodos , Espermidina/análise , Espermidina/química
7.
Food Chem ; 309: 125671, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31670129

RESUMO

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.


Assuntos
Fagopyrum/metabolismo , Glicoproteínas/química , Proteínas de Plantas/química , Sementes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Peso Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
8.
Yakugaku Zasshi ; 139(11): 1417-1425, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685738

RESUMO

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Stapf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae). The stems of Ephedra Herb contain greater than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). Despite its high effectiveness, Ephedra Herb exert several adverse effects, including palpitation, excitation, insomnia, and dysuria. Both the primary and adverse effects of Ephedra Herb have been traditionally believed to be mediated by these ephedrine alkaloids. However, our study found that several pharmacological actions of Ephedra Herb were not associated with ephedrine alkaloids. We prepared an ephedrine alkaloid-free Ephedra Herb extract (EFE) by eliminating ephedrine alkaloids from Ephedra Herb extract (EHE) using ion-exchange column chromatography. EFE exerted analgesic, anti-influenza, and anticancer activities in the same manner as EHE. Moreover, EFE did not induce adverse effects due to ephedrine alkaloids, such as excitation, insomnia, and arrhythmias, and showed no toxicity. Furthermore, we evaluated the safety of EFE in healthy volunteers. The number of adverse event cases was higher in the EHE-treated group than in the EFE-treated group, although the difference was not significant. Our evidence suggested that EFE was safer than EHE.


Assuntos
Medicamentos de Ervas Chinesas/efeitos adversos , Medicamentos de Ervas Chinesas/química , Ephedra/química , Idoso , Analgésicos , Antineoplásicos Fitogênicos , Antivirais , Cromatografia por Troca Iônica , Medicamentos de Ervas Chinesas/farmacologia , Efedrina/efeitos adversos , Efedrina/isolamento & purificação , Feminino , Humanos , Masculino , Pseudoefedrina/efeitos adversos , Pseudoefedrina/isolamento & purificação , Segurança
9.
Artigo em Inglês | MEDLINE | ID: mdl-31698243

RESUMO

In this study a comprehensive approach for determination of low molecular organophosphorus nerve agent markers - highly polar alkylphosphonic acids and much less polar alkyl methylphosphonic acids is presented. Accurate, sensitive and simultaneous determination of the nerve agent markers in human urine was performed by ion chromatography and tandem mass spectrometry using deuterated internal standards. Analysis of the urine extracts was conducted on an anion-exchanger based on poly(styrene-co-divinylbenzene) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer. The use of this type of column allowed achieving high values of retention factors for alkylphosphonic acids and alkyl methylphosphonic acids due to combination of anion-exchange and hydrophobic interactions between the analytes and the stationary phase of the column. Prior to the analysis, the urine samples were purified using anion-exchange cartridges for solid-phase extraction, and high recovery values were achieved for each analyte. The developed IC-MS/MS technique was validated for linearity, limit of detection, limit of quantification, precision and accuracy using two LC-MS/MS instruments. The proposed approach was successfully tested on the urine samples, provided by the Organisation for the Prohibition of Chemical Weapons in the frame of the 4th Biomedical Proficiency Test.


Assuntos
Cromatografia por Troca Iônica/métodos , Agentes Neurotóxicos/análise , Compostos Organofosforados/urina , Extração em Fase Sólida/métodos , Ânions , Biomarcadores/urina , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
10.
Artigo em Inglês | MEDLINE | ID: mdl-31731217

RESUMO

Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection. In this study, a newly developed 3 µm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 and 50 µg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 µg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.


Assuntos
Anticorpos Monoclonais , Subunidades de Imunoglobulinas , Espectrometria de Massas/métodos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Subunidades de Imunoglobulinas/análise , Subunidades de Imunoglobulinas/química , Subunidades de Imunoglobulinas/isolamento & purificação , Modelos Lineares , Modelos Moleculares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Anal Chim Acta ; 1087: 121-130, 2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31585560

RESUMO

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.


Assuntos
Proteínas Amiloidogênicas/análise , Príons/análise , Agregados Proteicos , Proteínas Amiloidogênicas/isolamento & purificação , Proteínas Amiloidogênicas/toxicidade , Linhagem Celular Tumoral , Cromatografia por Troca Iônica , Fracionamento por Campo e Fluxo , Humanos , Luz , Microscopia de Força Atômica , Tamanho da Partícula , Príons/isolamento & purificação , Príons/toxicidade , Espalhamento de Radiação
12.
Se Pu ; 37(9): 996-1003, 2019 Sep 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642305

RESUMO

A method of gradient elution ion chromatography with integrated pulsed amperometric detection for simultaneous determination of twenty amino acids and six carbohydrates in soy sauce was established. The effects of column temperature, pH value of solution, and standing time were studied, and then the suitable gradient elution conditions were found for the determination. In the conditions that the flow rate is 0.25 mL/min, pH value of solution is in the range of 5.2-6.7, temperature of column is 35℃, the linear relationships of the 26 components are good and the correlation coefficients are not less than 0.995. Except glutamine, leucine, isoleucine and methionine, the limits of detection (S/N=3) of the other 22 components are less than 0.03 mg/L. The recoveries of the 26 components in soy sauce are 84.2%-109% at the spiked levels of 0.20, 0.50, 2.00 mg/L, and the relative standard deviations (RSDs) are in the range of 2.7%-7.8%. The method is efficient, easy, sensitive, and accurate for simultaneous determination of amino acids and carbohydrates in soy sauce, and can provide an effective research technique for adulterated soy sauce.


Assuntos
Aminoácidos/análise , Carboidratos/análise , Alimentos de Soja/análise , Cromatografia por Troca Iônica
13.
World J Microbiol Biotechnol ; 35(10): 150, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31549229

RESUMO

This study aimed to characterize a powerful antifungal component from bacteria. Bacillus subtilis strain XB-1, which showed maximal inhibition of Monilinia fructicola, was isolated and identified, and an antifungal protein was obtained from it. Ammonium sulfate precipitation, ion exchange chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to purify and identify the proteins secreted by B. subtilis XB-1. Analyses revealed that purified fraction V had the strongest antifungal effect, with the largest pathogen inhibition zone diameter of 4.15 cm after 4 days (P < 0.05). This fraction showed a single band with a molecular weight of approximately 43 kDa in SDS-PAGE. Results from SDS-PAGE and liquid chromatography electrospray ionization tandem mass spectrometry analyses demonstrated that fraction V was likely a member of the chitosanase family. These results suggest that B. subtilis XB-1 and its antifungal protein may be useful in potential biocontrol applications.


Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Bacillus subtilis/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/farmacologia , Antifúngicos/química , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/química , Peso Molecular
14.
Biochemistry (Mosc) ; 84(8): 963-977, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522678

RESUMO

Cucumber target leaf spot (TLS) disease caused by Corynespora cassiicola has become one of the most important fungal foliar diseases of cultivated cucumbers. However, the defense mechanisms of cucumber plants (Cucumis sativus) against C. cassiicola are still poorly understood. Here, proteins from resistant cucumber plants were analyzed using iTRAQ (isobaric tags for relative and absolute quantification) method. A total of 286 differentially expressed proteins were identified (p < 0.05, ratio > 1.2 or < 0.83) 6 and 24 h after pathogen inoculation in the resistant cultivar Jinyou 38 (the data are available via ProteomeXchange; identifier, PXD012903). Some of the early responses to C. cassiicola infection were revealed, and four factors related to the resistance of cucumber plants to TLS were discovered. First, the proteomic approach revealed modulation of signaling pathways in resistant cucumber plants in response to C. cassiicola infection. Second, the plant immune system recognizes the pathogen and initiates expression of immune response proteins, including those related to plant defense, stress response, signal transduction, cell metabolism, and redox regulation. Third, C. cassiicola activates common stress response pathways; in particular, mildew resistance locus O (MLO) proteins were found to play a crucial role in the TLS prevention. Fourth, rapid activation of the carbohydrate and secondary metabolic pathways, modification and reinforcement of cell walls, and adjustment of the apoplastic environment to the highly stressful conditions were crucial in the cucumber resistance to TLS. Overall, our data contribute to the understanding of interactions between plants and their pathogens and provide new insight into molecular processes involved in the resistance of cucumber plants to disease.


Assuntos
Ascomicetos/patogenicidade , Cucumis sativus/metabolismo , Cucumis sativus/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/biossíntese , Proteínas de Ligação a Calmodulina , Parede Celular/metabolismo , Cromatografia por Troca Iônica , Bases de Dados de Proteínas , Resistência à Doença/fisiologia , Células Vegetais/metabolismo , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/isolamento & purificação , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas em Tandem , Terpenos/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-31476578

RESUMO

A baseline dip caused by the reduction of dissolved oxygen in samples has been a source of trouble in the analysis of major monosaccharides (galactose, glucose, mannose, and fructose) in the high-performance anion-exchange chromatography with pulsed amperometric detection system. This study attempted three different methods to remove the baseline dip from the resulting chromatograms, and among the approaches, sodium sulfite was found to act as the best oxygen scavenger. Clean chromatograms were obtained by adding at least 3 mg/mL sodium sulfite to samples, which removed the baseline dip and improved the accuracy of sugar analysis. Although sodium sulfite does not influence analytical sensitivity, it can cause a reduction of sugar retention; however, retention time can be recovered by washing with 200 mM sodium hydroxide solution. Results demonstrated that sodium sulfite is an effective means either to remove the baseline dip for low concentration analysis under 1 mg/L, or to separate the target sugar from the baseline dip by retention time rearrangement.


Assuntos
Cromatografia por Troca Iônica/métodos , Monossacarídeos/análise , Oxigênio/química , Sulfitos/química , Modelos Lineares , Monossacarídeos/química , Reprodutibilidade dos Testes
16.
J Chromatogr A ; 1604: 460487, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31488296

RESUMO

The goal of this study was to better understand the possibilities and limitations of modern cation exchange chromatography (CEX) columns for the separation of protein biopharmaceuticals (typically mAbs and related products). Several commercial and research columns consisting of a non-porous polymeric core particle with a thin hydrophilic coating and grafted ion-exchanger sulfonate groups, were compared. The impact of particle size, porosity and packing pressure on the separation of therapeutic proteins was evaluated in a systematic way. First, it was shown that the porosity of modern CEX columns depends on the applied conditions, and lower apparent porosity as well as increased column pressures were observed when using low ionic strength mobile phase (less than 0.01 M NaCl), due to swelling. Column pressure seemed to be dependent on the 1/dp3 to 1/dp5 relationships with particle size, depending on whether 0.3 M NaCl or pure water was used as mobile phase, respectively. Using 5 cm long columns packed with 2 or 2.5 µm particles could easily result in higher than 1000 bar pressure drops when the mobile phase ionic strength is low. Therefore, it is recommended that particle size not be decreased to below 2.5 µm so that technologies can remain compatible with the current state of ultra-high pressure (UHPLC) instrumentation. This recommendation is underscored by the fact that a decrease in particle size does not produce improved separations, since the particles are non-porous (no intra-particle diffusion nor resistance to mass transfer) and that large solutes follow an on-off (bind and elute) type retention mechanism. The only advantage of CEX columns packed with small particles is that they can provide more specific surface area per unit length of column, and thus facilitate higher throughput methods. In conclusion, it appears that there is no need to further decrease the particle size in CEX since decreasing their particle size may result in more drawbacks than benefits.


Assuntos
Produtos Biológicos/isolamento & purificação , Tamanho da Partícula , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica , Interações Hidrofóbicas e Hidrofílicas , Permeabilidade , Porosidade , Pressão
17.
J Chromatogr A ; 1608: 460410, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31395360

RESUMO

PEGylation of biological macromolecules is a well-established strategy to increase circulation half-life, decrease renal clearance and improve biocompatibility. PEGylation is a process in which polyethylene glycol (PEG) is covalently attached to a target molecule. The production of PEGylated biopharmaceuticals is usually executed by first producing and purifying the base molecule followed by the PEGylation reaction and purification of the modified molecule. Most PEGylated pharmaceuticals are produced by random PEGylation in batch mode and need to be purified as mainly the mono-PEGylated form is the desired drug product. In this work we propose a method to estimate the degree of PEGylation (DOP) of modified protein eluting from a chromatography column in near real-time. extended multiplicative signal correction (EMSC) is used in conjunction with asymmetric least squares (aaLS) to alleviate the influence of a salt gradient during ion exchange chromatography (IEX) on the spectral data. To convert the raw data obtained from spectral data to the actual DOP additional information obtained from off-line measurements is utilized. Once the signal correction is applied to in-line spectral data the DOP can be estimated without further use of off-line analytics. As the prerequisites for the application of this method are relatively easy to obtain it may also find use to speed up process development.


Assuntos
Produtos Biológicos/química , Química Farmacêutica/métodos , Cromatografia por Troca Iônica , Polietilenoglicóis/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Análise dos Mínimos Quadrados , Polietilenoglicóis/química
18.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1126-1127: 121742, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387012

RESUMO

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis.


Assuntos
Alcaloides/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Corydalis/química , Medicamentos de Ervas Chinesas/química , Alcaloides/química , Cátions , Cromatografia por Troca Iônica/instrumentação , Cromatografia de Fase Reversa/instrumentação
19.
Anal Chim Acta ; 1079: 220-229, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387715

RESUMO

We developed temperature-responsive mixed-mode columns packed with poly(N-isopropylacrylamide) (PNIPAAm)-modified beads and poly(2-acrylamido-2-methylpropane sulfonic acid) (PAMPS)-modified beads in various ratios. The PNIPAAm-modified silica beads and PAMPS-modified silica beads were prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropane sulfonic acid, respectively. We confirmed polymer modification of the silica beads by CHN elemental analysis, FTIR, zeta-potential measurements, and SEM. To determine the column separation efficiency, we examined the elution behaviors of cold medicine active ingredients and monoamines from each column. Analyte separation occurred on columns with PNIPAAm to PAMPS ratios of 1:20 and 1:10, whereas a column containing only PNIPAAm-modified beads did not retain the analytes. The analytes were retained on the columns through hydrophobic and electrostatic interactions with PNIPAAm and PAMPS, respectively. The separation performance improved with increasing column temperature because of dehydration of PNIPAAm and enhancement of hydrophobic interactions at elevated temperatures. The mixed-mode columns will be useful for separating basic bioactive compounds because the retention of analytes can be modulated by changing the column temperature and the composition.


Assuntos
Resinas Acrílicas/química , Resinas de Troca de Ânions/química , Cromatografia por Troca Iônica/instrumentação , Polímeros/química , Ácidos Sulfônicos/química , Alcaloides/análise , Aminas/análise , Cromatografia por Troca Iônica/métodos , Compostos Heterocíclicos de Anéis Fundidos/análise , Dióxido de Silício/química , Temperatura Ambiente
20.
EMBO J ; 38(21): e100847, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31433507

RESUMO

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.


Assuntos
Ânions/química , Cromatografia por Troca Iônica/métodos , Fosfopeptídeos/análise , Fosfoproteínas/análise , Proteoma/análise , Biologia Computacional , Células HeLa , Humanos , Espectrometria de Massas , Fosforilação
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