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1.
Food Chem ; 335: 127645, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738537

RESUMO

The dried Ganoderma lucidum (GL) has been widely used for its pharmacological properties and bioactive ganoderic acids (GAs). Herein, extraction procedures combining ultra-sonication and heating were optimized using response surface methodology based on four variables (antioxidant activity, anti-diabetic activity, total GAs content, and total polysaccharide content) and principal component analysis. The extraction of freeze-dried GL at temperatures between 64.2 and 70 °C for 1.2 h maximized the antioxidant activity and GA content, whereas the polysaccharide content and anti-diabetic activity were maximized by extraction between 66.8 and 70 °C for more than 2.8 h. Heat-dried GL extracted at 50 °C for 3 h provided the greatest anti-inflammatory activity against HaCaT cells by suppressing the response to inflammation related cytokines at mRNA levels. These results suggest that extraction conditions might be a limiting factor for target-oriented investigations, and optimized extraction methods may improve the potential effect and quality of harvested GL products.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Antioxidantes/isolamento & purificação , Fracionamento Químico/métodos , Hipoglicemiantes/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Reishi/química , Triterpenos/isolamento & purificação , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Linhagem Celular , Fracionamento Químico/instrumentação , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Triterpenos/química , Triterpenos/farmacologia
2.
Food Chem ; 340: 127918, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889209

RESUMO

The study was an attempt to compare batch and circulating processes for polyphenols extraction from pomelo peels by liquid-phase pulsed discharge (LPD) in order to assess the extraction efficiency of the two processes. Response surface methodology was used to optimize batch (8-12 kV discharge voltage, 30-50 mL/g liquid to solid ratio and 2-4 min extraction time) and circulating (8-12 kV discharge voltage, 30-50 mL/g liquid to solid ratio and 20-40 mL/min flow rate) extractions. The highest polyphenols yield was 2.50 ± 0.02% at 42.2 mL/g, 12 kV and 4 min in batch extraction, while circulating extraction produced the most polyphenols (2.42 ± 0.01%) at 43.7 mL/g, 10.4 kV and 27.6 mL/min. The results showed that batch extraction achieved much greater yields than circulating extraction with lower-cost equipment. Therefore, batch extraction was a promising technology for the separation of high value-added products from pharmaceuticals and fine chemicals.


Assuntos
Fracionamento Químico/métodos , Citrus/química , Frutas/química , Polifenóis/isolamento & purificação , Fracionamento Químico/instrumentação , Indústria de Processamento de Alimentos/instrumentação , Indústria de Processamento de Alimentos/métodos
3.
Food Chem ; 338: 128144, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33092004

RESUMO

A weak ion-exchange membrane (P-COOH) was synthesized by alkaline hydrolysis of a polyacrylonitrile nanofiber membrane prepared by electrospinning process. The P-COOH membrane was characterized for its physical properties and its application for purification of lysozyme from chicken egg white was investigated. The lysozyme adsorption efficiency of the P-COOH membrane operating in a stirred cell contactor (Millipore, Model 8010) was evaluated. The effects of key parameters such as the feed concentration, the rotating speed, the flow rate of feed and the operating pressure were studied. The results showed successful purification of lysozyme with a high recovery yield of 98% and a purification factor of 63 in a single step. The purification strategy was scaled-up to the higher feedstock loading volume of 32.7 and 70 mL using stirred cell contactors of Model 8050 and 8200, respectively. The scale-up processes achieved similar purification results, proving linear scalability of the purification technique adopted.


Assuntos
Fracionamento Químico/instrumentação , Clara de Ovo , Membranas Artificiais , Muramidase/isolamento & purificação , Nanofibras/química , Resinas Acrílicas/química , Adsorção , Animais , Troca Iônica , Muramidase/química
4.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
5.
J Chromatogr A ; 1620: 461004, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32143875

RESUMO

In the present study, a fast multiresidue method determining three novel fungicides fenpicoxamid, isofetamid, and mandestrobin in cereals was developed and validated for the first time using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Samples were extracted by QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology, and cleaned up using the disposable pipette extraction (DPX) tips containing primary secondary amine (PSA) and silica gel modified with zirconium oxide (Z-Sep) in less than 1 min. Linearity (r > 0.99) of three fungicides in the calibration range of 0.001-0.1 µg mL-1 was satisfactory. Mean recoveries (n = 15) from all matrices were between 84.8% and 100.3% as the corresponding intra-day and inter-day relative standard deviations (RSDs) were less than 10.6%. Limits of quantitation (LOQs) of all analytes in different matrices were defined at 0.01 mg kg-1. The results indicate this method can serve as a sensitive and rapid approach to monitoring contents of fenpicoxamid, isofetamid, and mandestrobin in cereals.


Assuntos
Acetamidas/análise , Cromatografia Líquida de Alta Pressão/métodos , Grão Comestível/química , Fungicidas Industriais/análise , Resíduos de Praguicidas/análise , Espectrometria de Massas em Tandem/métodos , Tiofenos/análise , Fracionamento Químico/instrumentação , Fungicidas Industriais/química , Fungicidas Industriais/isolamento & purificação , Lactonas/análise , Lactonas/química , Lactonas/isolamento & purificação , Resíduos de Praguicidas/química , Resíduos de Praguicidas/isolamento & purificação , Piridinas/análise , Piridinas/química , Piridinas/isolamento & purificação , Zircônio/química
6.
Food Chem ; 314: 126168, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951889

RESUMO

In this work, a kinetic model for protein extraction from Camellia oleifera seed cake using high voltage electrical discharge extraction (HVED) was built with discharge energy inputs as primary variables. The results showed that both the equilibrium yields and the mass transfer coefficient of HVED were highly dependent on the HVED specific energy input per pulse (kJ/kg). After linear and nonlinear fitting with five different basic functions, the best model satisfied the power function relationship through optimizing the influence of specific energy input per pulse on the equilibrium yields and the mass transfer coefficients. Based on the observations, a predictive model that correlates the energy input, mass of raw material and kinetics of HVED extraction was proposed. The validity of the predictive model was verified, and the observed deviation was found to be less than 10%. This could provide a model basis for optimization of HVED at different processing capacities.


Assuntos
Camellia/química , Fracionamento Químico/métodos , Técnicas Eletroquímicas/métodos , Extratos Vegetais/isolamento & purificação , Fracionamento Químico/instrumentação , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Cinética , Modelos Teóricos , Extratos Vegetais/química , Proteínas de Plantas/isolamento & purificação , Reprodutibilidade dos Testes , Sementes/química
7.
J Environ Sci Health B ; 55(1): 60-68, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31971076

RESUMO

This work reports the development of a very-simple-to-construct stir-bar extraction device so called "a dumbbell-shaped stainless steel stir-bar." The extraction device was assembled from a rolled up stainless steel net filled with an XAD-2 sorbent and a metal rod to allow the use of a magnetic stirrer during extraction. The dumbbell-shaped stainless steel stir-bar was used to extract diethyl phthalate (DEP), dibutyl phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) before analysis by a gas chromatograph equipped with an electron capture detector (GD-ECD). Under the optimal conditions, the developed method provided a good linearity from 10.0 to 1,000.0 ng mL-1 for all three compounds. Limits of detection and limits of quantification were 9.37 ± 0.29 ng mL-1 and 31.22 ± 0.95 ng mL-1 for DEP, 5.73 ± 0.31 ng mL-1 and 19.1 ± 1.0 ng mL-1 for DBP and 3.30 ± 0.06 ng mL-1 and 11.0 ± 0.19 ng mL-1 for DEHP, respectively. Good recoveries in the range of 81.89 ± 0.17 to 109.5 ± 2.0% were achieved when the method was used to extract phthalate esters in five instant noodle and two rice soup samples.


Assuntos
Fracionamento Químico/instrumentação , Análise de Alimentos/instrumentação , Oryza , Ácidos Ftálicos/isolamento & purificação , Aço Inoxidável , Fracionamento Químico/métodos , Cromatografia Gasosa , Dibutilftalato/isolamento & purificação , Dietilexilftalato/isolamento & purificação , Desenho de Equipamento , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Limite de Detecção , Ácidos Ftálicos/química , Reprodutibilidade dos Testes
8.
J Food Sci ; 85(3): 727-735, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31999367

RESUMO

Phycocyanin, a natural blue colorant, is typically extracted from liquid biomass of Arthrospira platensis, a blue-green algae called spirulina. In this study, we developed a scalable process to extract phycocyanin from dried spirulina biomass. First, we established the optimal ionic strength and pH for the extraction buffer. The results showed that a minimum ionic strength (>5 g/L NaCl) must be maintained to minimize the co-extraction of the green chlorophyll. The optimal pH of the phosphate buffer (100 mM) for phycocyanin extraction is 7.5; however, the pH should be immediately adjusted to 6.0 to 6.5 after the extraction to keep phycocyanin stable. Second, we also investigated three processing techniques, that is, high-pressure processing (HPP), pulsed electric field (PEF), and ultrasonication, to break the cell walls of spirulina and facilitate the release of phycocyanins into extraction buffers. HPP and PEF do not lead to the release of phycocyanin into the extraction buffer. However, ultrasonication breaks down the spirulina into fine particles and releases most of the phycocyanin, along with other impurities, immediately after the treatment. Last, it has been revealed that most of the phycocyanin can be extracted from biomass within 3 hr by phosphate buffer only (pH 7.5, 100 mM) at room temperature. It is concluded that there is no need to treat the rehydrated biomass solution by HPP, PEF, or ultrasonication due to the minimal benefits they brought for the extraction. Based on these results, we proposed an extraction process for the plant production of phycocyanin starting from dried spirulina biomass. PRACTICAL APPLICATIONS: Limited information can be found on the extraction of phycocyanin from dried spirulina biomass, especially how to better preserve the natural blue color of phycocyanin during extraction. We have investigated the method and presented a different view from previous processes. Pulsed electric field, high-pressure processing, and ultrasonication were employed to accelerate the extraction of phycocyanin from dried biomass. However, it was found that, unlike the extraction from live wet biomass, these techniques do not help with the extraction from dried biomass. Based on investigations, we have proposed a process that can be easily scaled up for the commercial production of phycocyanin from dried spirulina biomass.


Assuntos
Fracionamento Químico/métodos , Corantes de Alimentos/isolamento & purificação , Manipulação de Alimentos/métodos , Ficocianina/isolamento & purificação , Spirulina/química , Biomassa , Fracionamento Químico/instrumentação , Clorofila/análise , Clorofila/isolamento & purificação , Corantes de Alimentos/análise , Manipulação de Alimentos/instrumentação , Concentração de Íons de Hidrogênio , Ficocianina/análise , Spirulina/crescimento & desenvolvimento
9.
J Sep Sci ; 43(4): 799-807, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31769594

RESUMO

Hydrosoluble trehalose lipid (a biosurfactant) was employed for the first time as a green extraction solution to extract the main antioxidant compounds (geniposidic acid, chlorogenic acid, caffeic acid, and rutin) from functional plant tea (Eucommia ulmoides leaves). Single-factor tests and response surface methodology were employed to optimize the extraction conditions for ultrasound-assisted micellar extraction combined with ultra-high-performance liquid chromatography in succession. A Box-Behnken design (three-level, three-factorial) was used to determine the effects of extraction solvent concentration (1-5 mg/mL), extraction solvent volume (5-15 mL), and extraction time (20-40 min) at a uniform ultrasonic power and temperature. In consequence, the best analyte extraction yields could be attained when the trehalose lipid solution concentration was prepared at 3 mg/mL, the trehalose lipid solution volume was 10 mL and the extraction time was set to 35 min. In addition, the recoveries of the antioxidants from Eucommia ulmoides leaves analyzed by this analytical method ranged from 98.2 to 102%. These results indicated that biosurfactant-enhanced ultrasound-assisted micellar extraction coupled with a simple ultra-high-performance liquid chromatography method could be effectively applied in the extraction and analysis of antioxidants from Eucommia ulmoides leaf samples.


Assuntos
Antioxidantes/isolamento & purificação , Fracionamento Químico/métodos , Eucommiaceae/química , Lipídeos/química , Extratos Vegetais/isolamento & purificação , Trealose/química , Antioxidantes/análise , Fracionamento Químico/instrumentação , Cromatografia Líquida de Alta Pressão , Extratos Vegetais/análise , Folhas de Planta/química , Tensoativos/química , Chá/química , Ultrassom
10.
J Pharm Biomed Anal ; 177: 112879, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31542418

RESUMO

Globe artichoke (Cynara cardunculus var. scolymus L.) is not only used as a vegetable and ornamental plant, but also as important medicinal plant for the treatment of dyspeptic disorders. The European Pharmacopoeia describes a method for the quality assessment of dry artichoke leaves, which is time-consuming and requires huge amounts of organic solvents. In this study, an ultrasound-assisted extraction method was studied, which proved to be more efficient than the standard protocol of the European Pharmacopoeia, since it led to comparable results, was faster and easier to handle, and was more sustainable due to a reduced need for organic solvents.


Assuntos
Fracionamento Químico/métodos , Cynara scolymus/química , Flavonoides/isolamento & purificação , Hidroxibenzoatos/isolamento & purificação , Extratos Vegetais/análise , Fracionamento Químico/instrumentação , Química Farmacêutica/instrumentação , Química Farmacêutica/métodos , Estudos de Viabilidade , Metanol/química , Extratos Vegetais/química , Folhas de Planta/química , Solventes/química , Sonicação , Água/química
11.
J Food Sci ; 85(1): 150-156, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31877234

RESUMO

Fish-scale waste is rich in biocompatible hydroxyapatite (HAp). In the present study, an environmentally friendly method of extracting HAp from fish-scale waste was developed in an effort to promote environmental sustainability. Deep eutectic solvents (choline chloride/glycerol, 1/2) were used to extract HAp from bighead carp (Aristichthys nobilis) scales. A relatively high extraction rate of 47.67% ± 1.81% was obtained under optimum conditions (70 °C, a solid/liquid ratio of 1/15 g/g and a 2.5 hr extraction time). The obtained HAp was characterized and its purity was determined using Fourier transform infrared spectroscopy and X-ray diffraction, respectively. The chemical composition was performed by energy-dispersive X-ray spectrometry and inductively coupled plasma-optical emission spectroscopy. Its morphology and particle size were observed using scanning electron microscopy and particle size distribution analysis. Thermogravimetric analysis was used to determine its thermal stability. Blood compatibility was determined using a hemolytic test. The results showed that this extraction yielded HAp with the irregular morphology, the higher Ca/P ratio, good thermal stability, and blood compatibility, indicating that the proposed method is an excellent alternative for the improved utilization of fish scale waste. PRACTICAL APPLICATION: Biocompatible hydroxyapatite (HAp) was extracted from fish scale (FS) waste by using an environmentally friendly deep eutectic solvent. The optimized extraction and structure characterization of extracted HAp were investigated in this study. The results showed that the extracted HAp had the irregular morphology, the higher Ca/P ratio, good thermal stability, and blood compatibility, which indicated that the proposed method was an excellent alternative to improving the utilization of FS waste.


Assuntos
Fracionamento Químico/métodos , Durapatita/isolamento & purificação , Resíduos/análise , Escamas de Animais/química , Animais , Carpas , Fracionamento Químico/instrumentação , Durapatita/análise , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Solventes/química , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier
12.
Sci Rep ; 9(1): 16120, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695137

RESUMO

Byproducts such as orange peel have potential uses because of their bioactive compounds, which are important for their potential to reduce the risk factors of diseases caused by aging. The lack of effective techniques and the high levels of pollution produced by the conventional extraction of bioactive compounds using organic solvents have highlighted the need to enhance the 'green chemistry' trend. This study evaluates the use of ultrasound to extract bioactive compounds from orange peel. The antioxidant capacity, phenolic content, ascorbic acid, total carotenoids, and HPLC profile of phenolic compounds from orange peel extracts were obtained by a physicochemical evaluation. The results demonstrate that the optimal conditions for the ultrasound-assisted extraction of bioactive orange peel compounds were a power of 400 W, a time of 30 min, and 50% ethanol in water. These conditions were used to obtain a total carotenoid concentration of 0.63 mg ß-carotene/100 g, vitamin C concentration of 53.78 mg AA/100 g, phenolic concentration of 105.96 mg GAE/100 g, and antioxidant capacity of ORAC = 27.08 mM TE and TEAC = 3.97 mM TE. The major phenolic compound identified in all orange peel extracts was hesperidin, with a maximum concentration of 113.03 ± 0.08 mg/100 g.


Assuntos
Fracionamento Químico/métodos , Citrus sinensis/química , Química Verde/métodos , Extratos Vegetais/isolamento & purificação , Antioxidantes/análise , Antioxidantes/isolamento & purificação , Carotenoides/análise , Carotenoides/isolamento & purificação , Fracionamento Químico/instrumentação , Frutas/química , Química Verde/instrumentação , Fenóis/análise , Fenóis/isolamento & purificação , Extratos Vegetais/análise , Ultrassom/instrumentação , Ultrassom/métodos
13.
Analyst ; 144(23): 7032-7040, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31651914

RESUMO

Digital PCR is a powerful amplification method for absolute quantification of nucleic acids. The systems that integrated the nucleic acid extraction and amplification can reduce detection time, improve accuracy, and reduce labor costs. However, current nucleic acid extraction systems cannot be integrated well with integrated fluidic circuit (IFC) dPCR or droplet digital PCR chips perfectly and limit the application of digital PCR. In this study, a polytetrafluoroethylene (PTFE)-based nucleic acid extraction (PNE) system, which was able to achieve fully closed extraction for micro samples and was able to be integrated with IFC dPCR or droplet digital dPCR (ddPCR) chips perfectly is proposed. For this system, PTFE tubing with an inner diameter of 1 mm was used to load the reagents and superparamagnetic particles (PMPs) were used to extract nucleic acids. The system can extract nucleic acids from cells and blood in 5 minutes. Meanwhile, when nucleic acid extraction was completed, PNE was able to be directly combined with IFC dPCR or ddPCR chips without any intermediate steps. Therefore, the PNE system can realize sample-in-digital-answer-out. It will be highly useful in point-of-care (POC) and promote the development and application of dPCR.


Assuntos
Fracionamento Químico/métodos , DNA/análise , Técnicas Analíticas Microfluídicas/métodos , Reação em Cadeia da Polimerase/métodos , RNA/análise , Adsorção , Fracionamento Químico/instrumentação , DNA/isolamento & purificação , Células Hep G2 , Humanos , Dispositivos Lab-On-A-Chip , Fenômenos Magnéticos , Técnicas Analíticas Microfluídicas/instrumentação , Testes Imediatos , Politetrafluoretileno/química , RNA/isolamento & purificação
14.
Anal Chem ; 91(16): 10458-10466, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31373797

RESUMO

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.


Assuntos
Antifibrinolíticos/isolamento & purificação , Bioensaio , Cromatografia Líquida/métodos , Fibrinolíticos/isolamento & purificação , Nanotecnologia/métodos , Peptídeo Hidrolases/isolamento & purificação , Animais , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cromatografia Líquida/instrumentação , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Nanotecnologia/instrumentação , Peptídeo Hidrolases/análise , Venenos de Serpentes/química , Serpentes/metabolismo
15.
Curr Microbiol ; 76(11): 1247-1255, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31375861

RESUMO

Salmonella enterica serovar typhimurium (S. typhimurium) causes food poisoning in human and animals. Its infection rate is the highest among all salmonella serotypes. Metabolomics is a potential way to study the pathogenesis of S. typhimurium via analysis of various small molecular substances. Due to the lack of a uniform protocol for the extraction of metabolites, we evaluated five commonly used extraction methods including cold methanol (CM), hot ethanol (HE), chloroform-methanol cocktail (CMC), perchloric acid (PCA), and alkali (AL) for their efficacy in extracting the intracellular metabolites of S. typhimurium. Samples were quenched in 60% methanol at - 40 °C, and then the five methods were used to extract the metabolites. After derivatization, all samples were analyzed on a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). Our results suggest that CM and HE extraction methods provide the best compromise allowing identification of 98 and 95 metabolites in a single analysis. For targeted metabolome analysis, the optimal extraction method for alcohols and organic acids is HE. CMC preferentially extracted lipid metabolites. PCA is suitable for extraction of small molecular carbohydrates. The optimal extraction method for macromolecular carbohydrates is the CM method.


Assuntos
Fracionamento Químico/métodos , Salmonella typhimurium/química , Fracionamento Químico/instrumentação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Metabolômica , Salmonella typhimurium/metabolismo , Espectrometria de Massas em Tandem
16.
Methods Mol Biol ; 2044: 69-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432407

RESUMO

Data-independent acquisition (DIA) is becoming more prominent as a method for comprehensive proteomic analysis of clinical samples due to its ability to acquire essentially all fragment ion spectra in a single LC-ESI-MS/MS experiment. Since the direct correlation between a precursor and its fragment ions is lost when acquiring all ions in a defined m/z range, one data analysis strategy is using so-called peptide spectral libraries. These are usually generated by measuring similar biological samples in data-dependent (DDA) mode. The peptide spectral library content is a major limitation for the successful identification from DIA data. This is because a fragment ion spectrum from the sample can only be matched, and thus identified, when it is present in the peptide spectral library. In order to enhance peptide spectral library size, the sample for generating the peptide spectral library can be subjected to extended separation strategies prior to DDA. These strategies are of special relevance for biological samples containing a few very high-abundant proteins, such as CSF, as they enlarge the identification of low-abundant proteins. In instances of CSF separation, suitable methods include the 1D SDS-PAGE of proteins and high-pH reversed-phase peptide fractionation. Both methods are based on different protein/peptide characteristics, are complementary with one another, and are inexpensive and easy to establish. Ideally, DDA spectra from samples generated with both methods combine to achieve a comprehensive spectral library.


Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Fracionamento Químico/métodos , Peptídeos/líquido cefalorraquidiano , Peptídeos/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/química , Fracionamento Químico/instrumentação , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Íons/líquido cefalorraquidiano , Íons/química , Biblioteca de Peptídeos , Peptídeos/química , Proteólise , Proteômica , Software , Espectrometria de Massas em Tandem
17.
Methods Mol Biol ; 2044: 81-110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432408

RESUMO

Proteomics is an indispensable tool for disease biomarker discovery. It is widely used for the analysis of biological fluids such as cerebrospinal fluid (CSF), blood, and saliva, which further aids in our understanding of disease incidence and progression. CSF is often the biospecimen of choice in case of intracranial tumors, as rapid changes in the tumor microenvironment can be easily assessed due to its close proximity to the brain. On the contrary studies comprising of serum or plasma samples do not truly reflect the underlying molecular alterations due to the presence of protective blood-brain barrier. We have described in here the detailed workflows for two advanced proteomics techniques, namely, 2D-DIGE (two-dimensional difference in-gel electrophoresis) and iTRAQ (isobaric tag for relative and absolute quantitation), for CSF analysis. Both of these techniques are very sensitive and widely used for quantitative proteomics analysis.


Assuntos
Neoplasias Encefálicas/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Fracionamento Químico/métodos , Glioma/líquido cefalorraquidiano , Proteômica/métodos , Neoplasias Encefálicas/química , Proteínas do Líquido Cefalorraquidiano/química , Fracionamento Químico/instrumentação , Glioma/química , Humanos , Espectrometria de Massas , Proteoma/química , Proteoma/metabolismo , Proteoma/normas , Proteômica/normas , Software , Coloração e Rotulagem/métodos , Microambiente Tumoral/genética , Eletroforese em Gel Diferencial Bidimensional/métodos , Fluxo de Trabalho
18.
Methods Mol Biol ; 2044: 129-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432411

RESUMO

Human cerebrospinal fluid (CSF) is a sample of choice in the study of brain disorders. This biological fluid circulates in the brain and the spinal cord and contains tissue-specific proteins, indicative of health and disease conditions. Despite its potential as a valid source of biological markers, CSF remains largely understudied as compared to blood, in particular due to its more invasive way of sampling.Challenges remain when performing proteomic analysis in clinical research studies. State-of-the-art mass spectrometry (MS) enables deep characterization of the human proteome. But some technical limitations are cardinal to be addressed, such as the capacity to routinely analyze large cohorts of samples. Importantly, a trade-off still needs to be made between the proteome coverage depth and the number of measured samples. In this context, we developed a scalable automated proteomic pipeline for the analysis of CSF. Because of its versatility, this workflow can be adapted to accommodate proteome coverage and/or sample throughput. It allows us to prepare and quantitatively analyze hundreds to thousands of CSF samples; it can also allow identification of more than 3000 proteins in a CSF sample when coupled with isoelectric focusing fractionation.In this chapter, we describe an end-to-end pipeline for the proteomic analysis of CSF. The main steps of the sample preparation comprise spiking of a standard, protein digestion, isobaric labeling, and purification; these are performed in a 96-well plate format enabling automation. Depending on the targeted depth of the CSF proteome, optional analytical steps can be included, such as the removal of abundant proteins and sample pre-fractionation. Liquid chromatography tandem MS as well as data processing and analysis complete the pipeline.


Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Proteoma/análise , Proteômica/métodos , Alquilação , Automação , Biomarcadores/líquido cefalorraquidiano , Encefalopatias/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/metabolismo , Fracionamento Químico/instrumentação , Cromatografia Líquida/métodos , Humanos , Proteólise , Proteoma/metabolismo , Software , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho
19.
Methods Mol Biol ; 2044: 155-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432412

RESUMO

Cerebrospinal fluid (CSF) is in direct contact with the brain and represents a valuable source of mediators that reflect metabolic processes occurring in the central nervous system (CNS). In this sense, mass spectrometry (MS) methods have proven to be sensitive in quantifying the proteomic profiles of CSF, therefore being able to detect biomarker candidates for neurological disorders. In particular, a key development has been the use of multiplexing technologies to easily identify and quantify complex protein mixtures. This chapter describes a workflow suitable for the analysis of CSF proteome using isobaric labeling coupled to strong cation-exchange chromatography fractionation for its potential use as a biomarker discovery platform. In this case, the isobaric tags for relative and absolute quantitation (iTRAQ) label all proteins in a sample via free amines at the N-terminus and on the side chain of lysine residues. Then, the labeled samples are pooled and chromatographically fractionated. These fractions with the pooled samples are afterward analyzed by tandem mass spectrometry (MS/MS), and proteins are quantified by the relative intensities of the reporter ions in the MS/MS spectra, simultaneously obtaining the amino acid sequence. This method complements the neuroproteomic toolbox to identify new protein biomarkers not only for the early clinical diagnosis and disease staging of CNS-related disorders but also to elucidate the molecular mechanisms related to the pathophysiology of these symptoms.


Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteoma/metabolismo , Proteômica/métodos , Cátions/química , Proteínas do Líquido Cefalorraquidiano/sangue , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia Líquida , Humanos , Proteólise , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Fluxo de Trabalho
20.
Anal Chem ; 91(18): 11848-11855, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31411020

RESUMO

The extraction of bioanalytes is the first step in many diagnostic and analytical assays. However, most bioanalyte extraction methods require extensive dilution-based washing processes that are not only time-consuming and laborious but can also result in significant sample loss, limiting their applications in rare sample analyses. Here, we present a method that enables the efficient extraction of multiple different bioanalytes from rare samples (down to 10 cells) without washing-centrifugation-assisted immiscible fluid filtration (CIFF). CIFF utilizes centrifugal force to drive the movement of analyte-bound glass microbeads from an aqueous sample into an immiscible hydrophobic solution to perform an efficient, simple, and nondilutive extraction. The method can be performed using conventional polymerase chain reaction (PCR) tubes with no requirement of specialized devices, columns, or instruments, making it broadly accessible and cost-effective. The CIFF process can effectively remove approximately 99.5% of the aqueous sample in one extraction with only 0.5% residual carryover, whereas a traditional "spin-down and aspirate" operation results in a higher 3.6% carryover. Another unique aspect of CIFF is its ability to perform two different solid-phase bioanalytes extractions simultaneously within a single vessel without fractionating the sample or performing serial extractions. Here we demonstrate efficient mRNA and DNA extraction from low-input samples (down to 10 cells) with slightly higher to comparable recovery compared to a traditional column-based extraction technique and the simultaneous extraction of two different proteins in the same tube using CIFF.


Assuntos
Centrifugação/métodos , Fracionamento Químico/métodos , DNA/isolamento & purificação , Filtração/métodos , RNA Mensageiro/isolamento & purificação , Fracionamento Químico/instrumentação , Humanos , Reação em Cadeia da Polimerase/instrumentação , Proteínas/isolamento & purificação , Propriedades de Superfície , Células THP-1
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