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1.
J Chromatogr A ; 1626: 461319, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797815

RESUMO

The aim of this study is to model, describe and predict the mass transfer of IgG as a function of the agarose concentration in the protein A stationary phase, taking into account the influence of adsorption on the pore size. Therefore, particle size distribution, bed and bead porosities were examined by light microscopy, pressure-flow behavior and iSEC. Three agarose protein A stationary phases (2 wt%, 4 wt%, 6 wt%) were investigated. The pore size decreased from 116 nm for 2 wt% to 54 nm for 6 wt% and the porosity for the target molecule IgG was reduced by 25%. A shrinking core model approach was used to assess the influence of IgG adsorption on the pore size of the stationary phase and the diffusivity of IgG. Due to IgG adsorption, the pore diameter reduced by 24 nm, which is approximately two times its hydrodynamic diameter. Effective pore diffusivities of IgG were obtained by fitting the general rate model to breakthrough curves. They were in the range between 3.96·10-12m2/s and 6.5·10-12m2/s, decreasing as the agarose concentration increased. The DBC1% has a maximum for the 4 wt% agarose gel, showing optimal tradeoffs between accessibility, specific surface and diffusive mass transfer for IgG. A simple geometrical model was developed to describe the change in pore and filament diameters due to adsorption. The diffusion measured in protein A agarose beads can be described by a modification of the Ogston model. This enables the diffusion measured in protein A agarose networks to be predicted.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Sefarose/química , Adsorção , Cromatografia em Gel , Difusão , Imunoglobulina G/metabolismo , Tamanho da Partícula , Porosidade , Ligação Proteica , Proteína Estafilocócica A/metabolismo
2.
Nat Protoc ; 15(8): 2341-2386, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690956

RESUMO

Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of protein complexes on a proteome-wide scale is technically challenging. We have recently extended the targeted proteomics rationale to the level of native protein complex analysis (complex-centric proteome profiling). The complex-centric workflow described herein consists of size exclusion chromatography (SEC) to fractionate native protein complexes, data-independent acquisition mass spectrometry to precisely quantify the proteins in each SEC fraction based on a set of proteotypic peptides and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler (https://github.com/CCprofiler/CCprofiler). Complex-centric proteome profiling captures most proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to cultured cells and can potentially also be adapted to primary tissue and does not require any genetic engineering of the respective sample sources. At present, it requires ~8 d of wet-laboratory work, 15 d of mass spectrometry measurement time and 7 d of computational analysis.


Assuntos
Cromatografia em Gel , Espectrometria de Massas , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteômica/métodos , Células HEK293 , Humanos
3.
PLoS One ; 15(7): e0236508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726333

RESUMO

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.


Assuntos
Produtos Biológicos/sangue , Proteínas Sanguíneas/análise , Vesículas Extracelulares/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Cromatografia em Gel , Etanercepte/sangue , Etanercepte/uso terapêutico , Vesículas Extracelulares/química , Humanos , Imunoglobulina G/análise , Interleucina-8/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
4.
Toxicon ; 184: 94-98, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32533959

RESUMO

The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40-55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction.


Assuntos
Venenos de Cnidários/análise , Cubomedusas , Animais , Sobrevivência Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Miócitos Cardíacos , Nematocisto
5.
Chemosphere ; 258: 127278, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32554006

RESUMO

Syringe filters are widely used for sample pretreatments in laboratories. This study found that, surprisingly, these filters can leak dissolved organic carbon (DOC) that can potentially serve as precursors of disinfection by-products (DBPs). Nine common types of syringe filters were assessed. The results showed that the DOC of ultrapure water increased after syringe filtration. The DOC shed from filter membranes was characterized, whose spectra showed that the main compounds exhibited a low apparent molecular weight. Five classes of DBPs were investigated including trihalomethanes, haloacetaldehydes, haloacetonitriles, haloacetamides and halonitromethanes, among which trichloromethane (TCM), dichloroacetaldehyde (DCAL), trichloroacetaldehyde (TCAL), dichloroacetonitrile (DCAN), and trichloronitromethane (TCNM) were principally detected. The DBP formation was affected by chlorination time and membrane types. In general, the use of the poly vinylidene fluoride membrane resulted in the highest formation of TCM and TCAL, whereas nylon and mixed cellulose esters membranes contributed significantly to the formation of DCAN and TCNM, respectively. The shedding DOC and the formation of TCM, DCAL and TCAL from filter membranes were mitigated effectively by pre-washing; however, the contribution of membrane leakage to DCAN and TCNM formation was still notable, even with a pre-wash volume of 50 mL. When unwashed syringe filters were used for a real water sample, the DBP formation increased by up to 73.2% compared to the pre-washed ones; particularly for TCNM it was always over 15%. Therefore, for better quality control in laboratories, more attention should be paid to the syringe filters during sample pre-treatments, particularly when DBP formation is being investigated.


Assuntos
Desinfetantes/análise , Desinfetantes/química , Desinfecção/métodos , Membranas Artificiais , Acetonitrilos/análise , Acetonitrilos/química , Clorofórmio/análise , Clorofórmio/química , Cromatografia em Gel , Filtração/instrumentação , Filtração/métodos , Halogenação , Hidrocarbonetos Clorados/análise , Hidrocarbonetos Clorados/química , Peso Molecular , Trialometanos/análise , Trialometanos/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Purificação da Água/métodos
6.
Food Chem ; 330: 127319, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569936

RESUMO

The influence of fresh egg white (EW) addition on the quality characteristics and protein aggregation in oat noodles containing wheat flour and gluten was studied. EW addition decreased cooking loss and increased cooking time of 70% oat noodles. The hardness, chewiness, tensile force and tensile distance improved significantly. A smooth surface and continuous protein network were observed by scanning electron microscopy (SEM) after adding EW. After cooking, the peak area in SE-HPLC profile of 70% oat noodles with EW decreased obviously. The extractabilities of protein in sodium dodecyl sulfate containing medium (SDSEP) of cooked wheat and oat noodles under non-reducing condition were lower than those of samples under reducing condition. The protein bands changes in SDS-PAGE profiles showed that EW could induce disulfide cross-linking of proteins in noodles. EW addition promoted proteins interaction and improved the cooking and texture properties of oat noodles.


Assuntos
Avena , Clara de Ovo/química , Farinha , Qualidade dos Alimentos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Culinária , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Farinha/análise , Indústria de Processamento de Alimentos , Glutens , Dureza , Microscopia Eletrônica de Varredura , Agregados Proteicos , Triticum
7.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478746

RESUMO

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.


Assuntos
Aminopeptidases/química , Cristalografia por Raios X , Multimerização Proteica , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Aminopeptidases/isolamento & purificação , Aminopeptidases/metabolismo , Cromatografia em Gel , Cristalização , Recombinação Homóloga , Modelos Moleculares
8.
J Chromatogr A ; 1621: 461079, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32360061

RESUMO

Capto™ Core 700 is a core-shell chromatographic support with an adsorbing core contained within an inert shell layer designed to purify larger biomolecules and bioparticles in a flow-through mode. The present study aims to characterize the structure and functional properties of this resin using bovine serum albumin (BSA, Mr~65 kDa) and thyroglobulin (Tg, Mr~660 kDa) as model impurity proteins. The functionalized adsorbing core and the inert shell have the same fibrous structure typical of agarose-based beads. The resin average bead size is 90.7 µm with a range of 50-130 µm, the shell thickness is 4.18 µm with a range of 3-6 µm and a standard deviation of 0.55 µm, and the pore radius, obtained by inverse size exclusion chromatography, is 50.4 ± 1.3 nm. Both proteins present highly favorable binding isotherms with maximum binding capacities of 55 and 105 mg/mL of total bead volume for BSA and Tg, respectively. The addition of 500 mM NaCl reduces the binding capacity by less than 50%, showing the ability of the resin to operate at high salt conditions. For both proteins, the effective pore diffusivity in the core is smaller than in the shell due to additional hindrance by bound protein in the core area. Effective pore diffusivities values in the core are 1.6 × 10-7 and 0.16 × 10-7 cm2/s for BSA and Tg, respectively. The DBC10% at 2 min residence time are 24 and 2 mg/mL for BSA and Tg, respectively. This study provides qualitative and quantitative information about Capto™ Core 700 resin. This information could be used to predict and optimize the purification of large biomolecules and bioparticle in route to the establishment of more effective downstream processes.


Assuntos
Cromatografia em Gel/métodos , Tamanho da Partícula , Adsorção , Animais , Bovinos , Resinas Sintéticas/química , Soroalbumina Bovina/química , Temperatura , Tireoglobulina/química
9.
Food Chem ; 321: 126692, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32251923

RESUMO

In this work, we describe for the first time the presence of selenoprotein P in human breast milk. To this end, a novel analytical method has been developed based on a two-dimensional column switching system, which consisted of three size exclusion columns and one affinity column coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method combines the accurate quantification of selenoproteins and selenometabolites by species unspecific isotopic dilution ICP-MS, with unequivocal identification by quadrupole-time-of-flight mass spectrometry. Several selenopeptides, which contain the amino acid selenocysteine (U, SeCys), were identified after tryptic digestion followed by their separation. The results reveal that the relative selenium concentration in colostrum follows the order: glutathione peroxidase (GPX) ≈ selenoprotein P (SELENOP) > selenocystamine (SeCA) > other selenometabolites (SeMB), in contrast with previously published papers (GPX > SeCA > selenocystine > selenomethionine). A mean concentration of 20.1 ± 1.0 ng Se g-1 as SELENOP (1.45 µg SELENOP/g) was determined in colostrum (31% of total selenium).


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Leite Humano/química , Selenoproteína P/análise , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão/instrumentação , Feminino , Glutationa Peroxidase/metabolismo , Humanos , Selênio/análise , Selenocisteína/análise , Selenocisteína/química , Selenometionina/análise , Selenoproteínas/análise
10.
J Chromatogr A ; 1621: 461064, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32336499

RESUMO

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.


Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Tamanho da Partícula , Proteínas/análise , Proteínas/isolamento & purificação , Água/química
11.
J Chromatogr A ; 1620: 461011, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284152

RESUMO

Liquid-liquid partition chromatography has been used for many years as a model and teaching introduction to column chromatography. However, the partition model does not describe separations on bonded phases with porous supports particularly well, especially regarding the thermodynamics controlling solute distribution. Further difficulties arise when more than one mechanism is involved in solute retention. Nomenclature is not perfectly aligned with the underlying thermodynamic descriptors and is inconsistently applied over various chromatographic techniques. Presented here is a general description of retention that spans partition, size exclusion, and hydrodynamic separation processes, and is then applied to bonded-phase separations on porous supports. The model provides a general description applicable to adsorption, reversed-phase, hydrophilic interaction, size-exclusion, hydrodynamic chromatography, and any combination of these techniques including liquid chromatography at the critical condition. Further expansion to include retention by ion-exchange and field-flow fractionation appears to be possible. Recommendations on retention factor definition and evaluation are given.


Assuntos
Cromatografia em Gel , Hidrodinâmica , Adsorção , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Porosidade
12.
Water Res ; 174: 115652, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32135428

RESUMO

Assays based on the polymerase chain reaction (PCR) are widely applied to quantify enteric viruses in aquatic environments to study their fates and potential infection risks. However, inhibitory substances enriched by virus concentration processes can result in inaccurate quantification. This study aimed to find a method for improving virus quantification by mitigating the effects of inhibitory environmental concentrates, using previous knowledge of the properties of the inhibitory substances. Performances of anion exchange resins, gel filtration, and a hydrophobic resin (DAX-8) were comparatively evaluated using poliovirus and its extracted RNA spiked into humic acid solutions. These solutions served as good representatives of the inhibitory environmental concentrates. A sequential treatment using DAX-8 resin and gel filtration produced the most favorable results, i.e., low virus losses that were stable and a reduced inhibitory effect. Furthermore, the sequential treatment was applied to another set of 15 environmental concentrates. Without the sequential treatment, serious underestimation (>4.0 log10 to 1.1 log10) of a molecular process control (murine norovirus) was measured for eight samples. With the treatment, the control was detected with <1.0 log10 underestimation for all samples. The treatment improved the quantification of seven types of indigenous viruses. In summary, the sequential treatment is effective in improving the viral quantification in various of environmental concentrates.


Assuntos
Norovirus , Vírus , Animais , Cromatografia em Gel , Substâncias Húmicas , Camundongos , Reação em Cadeia da Polimerase , RNA Viral
13.
Food Chem ; 319: 126534, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32193058

RESUMO

The antioxidant peptides extracted from duck plasma hydrolysate (DPH) was investigated. The antioxidant activity of DPH, which was isolated and purified via ultrafiltration, size exclusion chromatography, and reversed-phase high-performance liquid chromatography, was evaluated using its free radical scavenging ability. Nano-liquid chromatography-tandem mass spectrometry was conducted to identify the DPH fractions with the highest antioxidant ability. Seven novel peptides: LDGP, TGVGTK, EVGK, RCLQ, LHDVK, KLGA, and AGGVPAG (400.43, 561.63, 431.48, 260.14, 610.71, 387.47, and 527.57 Da, respectively) were identified and synthesized using a solid-phase peptide produce to evaluate their antioxidant activities. Of these, EVGK exhibited the highest Fe2+ chelating ability (16.35%), and RCLQ presented the highest reducing power, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl scavenging rate (0.62, 274.83 mM TE/mg, and 95.12%, respectively). Our results indicated that DPH possessed antioxidant capabilities and could be used to obtain antioxidant peptides, thus adding economic value to duck blood.


Assuntos
Antioxidantes/química , Proteínas Sanguíneas/química , Patos , Peptídeos/química , Animais , Antioxidantes/isolamento & purificação , Compostos de Bifenilo , Proteínas Sanguíneas/isolamento & purificação , Cromatografia em Gel , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/química
14.
Int J Nanomedicine ; 15: 1253-1266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32161456

RESUMO

Purpose: This study aimed to develop a chelate-free radiolabeled nanoparticle platform for simultaneous positron emission tomography (PET) and magnetic resonance (MR) imaging that provides contrast-enhanced diagnostic imaging and significant image quality gain by integrating the high spatial resolution of MR with the high sensitivity of PET. Methods: A commercially available super-paramagnetic iron oxide nanoparticle (SPION) (Feraheme®, FH) was labeled with the [89Zr]Zr using a novel chelate-free radiolabeling technique, heat-induced radiolabeling (HIR). Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and radio-thin layer chromatography (radio-TLC). Characterization of the non-radioactive isotope 90Zr-labeled FH was performed by transmission electron microscopy (TEM). Simultaneous PET-MR phantom imaging was performed with different 89Zr-FH concentrations. The MR quantitative image analysis determined the contrast-enhancing properties of FH. The signal-to-noise ratio (SNR) and full-width half-maximum (FWHM) of the line spread function (LSF) were calculated before and after co-registering the PET and MR image data. Results: High RCY (92%) and RCP (98%) of the [89Zr]Zr-FH product was achieved. TEM analysis confirmed the 90Zr atoms adsorption onto the SPION surface (≈ 10% average radial increase). Simultaneous PET-MR scans confirmed the capability of the [89Zr]Zr-FH nano-platform for this multi-modal imaging technique. Relative contrast image analysis showed that [89Zr]Zr-FH can act as a dual-mode T1/T2 contrast agent. For co-registered PET-MR images, higher spatial resolution (FWHM enhancement ≈ 3) and SNR (enhancement ≈ 8) was achieved at a clinical dose of radio-isotope and Fe. Conclusion: Our results demonstrate FH is a highly suitable SPION-based platform for chelate-free labeling of PET tracers for hybrid PET-MR. The high RCY and RCP confirmed the robustness of the chelate-free HIR technique. An overall image quality gain was achieved compared to PET- or MR-alone imaging with a relatively low dosage of [89Zr]Zr-FH. Additionally, FH is suitable as a dual-mode T1/T2 MR image contrast agent.


Assuntos
Imagem por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Cromatografia em Gel , Cromatografia em Camada Delgada , Meios de Contraste/química , Óxido Ferroso-Férrico/química , Humanos , Nanopartículas de Magnetita/uso terapêutico , Imagens de Fantasmas , Radioisótopos/química , Razão Sinal-Ruído , Zircônio/química
15.
PLoS One ; 15(3): e0224002, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191706

RESUMO

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.


Assuntos
Reprodutibilidade dos Testes , Esferoides Celulares/metabolismo , Células CACO-2 , Varredura Diferencial de Calorimetria , Técnicas de Cultura de Células , Cromatografia em Gel , Células HT29 , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
16.
Artigo em Inglês | MEDLINE | ID: mdl-32145639

RESUMO

Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (Tm1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest Tm1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and Tm1, three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery.


Assuntos
Vírus da Febre Aftosa/química , Substâncias Macromoleculares/química , Adsorção , Cromatografia em Gel , Cromatografia por Troca Iônica , Humanos , Concentração de Íons de Hidrogênio , Transição de Fase , Propriedades de Superfície , Temperatura de Transição
17.
J Chromatogr A ; 1620: 460991, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115234

RESUMO

We present relationships between the multiscale structure and the separation properties of size exclusion chromatography (SEC) columns. Physical bed reconstructions of wall and bulk regions from a 2.1 mm i.d. column packed with fully porous 1.7 µm bridged-ethyl hybrid (BEH) particles, obtained by focused ion-beam scanning electron microscopy, serve as geometrical models for the packing microstructure in wall and central regions of a typical narrow-bore SEC column. In addition, the intraparticle mesopore space morphology of the BEH particles is reconstructed using electron tomography, to ultimately construct a realistic multiscale model of the bed morphology from mesopore level via interparticle macropore space to transcolumn scale. Complemented by the results of eddy dispersion simulations in computer-generated bulk packings, relationships between packing microstructure and transchannel, short-range interchannel, as well as transcolumn eddy dispersion are used to analyze the fluid dynamics in the interparticle macropore space of the model. Further, we simulate hindered diffusion and accessible porosity for passive, finite-size tracers in the intraparticle mesopore space, to finally determine the effective particle and bed diffusion coefficients of these tracers in the hierarchical (macro-mesoporous) bed. Retention and transport properties of polystyrene standards with hydrodynamic diameters from 5 to 95 Å in tetrahydrofuran are subsequently predicted without introducing bias from arbitrary models. These properties include the elution volumes of the polystyrene standards, the global peak capacity (over the entire separation window), and the rate of peak capacity at any fixed elution volume. Optimal flow rates yielding maximal global peak capacity and a nearly uniform rate of peak capacity over the entire separation window are close to 0.04 and 0.20 mL/min, respectively. SEC column performance obtained for fully porous and superficially porous particles is compared by varying the core-to-particle diameter ratio ρ from 0 to 0.95. Because the separation window is narrowing more rapidly than the rate of peak capacity is growing with increasing ρ, core-shell particles always provide smaller global peak capacity; they still can be advantageous but only for simple sample mixtures. The presented morphology-performance approach holds great promise for method development in SEC.


Assuntos
Cromatografia em Gel/métodos , Cromatografia em Gel/normas , Difusão , Tomografia com Microscopia Eletrônica , Furanos/química , Microscopia Eletrônica de Varredura , Poliestirenos/normas , Porosidade
18.
Int J Nanomedicine ; 15: 31-47, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021163

RESUMO

Purpose: Using our chelate-free, heat-induced radiolabeling (HIR) method, we show that a wide range of metals, including those with radioactive isotopologues used for diagnostic imaging and radionuclide therapy, bind to the Feraheme (FH) nanoparticle (NP), a drug approved for the treatment of iron anemia. Material and methods: FH NPs were heated (120°C) with nonradioactive metals, the resulting metal-FH NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), and r1 and r2 relaxivities obtained by nuclear magnetic relaxation spectrometry (NMRS). In addition, the HIR method was performed with [90Y]Y3+, [177Lu]Lu3+, and [64Cu]Cu2+, the latter with an HIR technique optimized for this isotope. Optimization included modifying reaction time, temperature, and vortex technique. Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and thin-layer chromatography (TLC). Results: With ICP-MS, metals incorporated into FH at high efficiency were bismuth, indium, yttrium, lutetium, samarium, terbium and europium (>75% @ 120 oC). Incorporation occurred with a small (less than 20%) but statistically significant increases in size and the r2 relaxivity. An improved HIR technique (faster heating rate and improved vortexing) was developed specifically for copper and used with the HIR technique and [64Cu]Cu2+. Using SEC and TLC analyses with [90Y]Y3+, [177Lu]Lu3+ and [64Cu]Cu2+, RCYs were greater than 85% and RCPs were greater than 95% in all cases. Conclusion: The chelate-free HIR technique for binding metals to FH NPs has been extended to a range of metals with radioisotopes used in therapeutic and diagnostic applications. Cations with f-orbital electrons, more empty d-orbitals, larger radii, and higher positive charges achieved higher values of RCY and RCP in the HIR reaction. The ability to use a simple heating step to bind a wide range of metals to the FH NP, a widely available approved drug, may allow this NP to become a platform for obtaining radiolabeled nanoparticles in many settings.


Assuntos
Óxido Ferroso-Férrico/química , Marcação por Isótopo/métodos , Nanopartículas/química , Radioisótopos/química , Quelantes/química , Cromatografia em Gel , Radioisótopos de Cobre/química , Difusão Dinâmica da Luz , Lutécio/química , Espectroscopia de Ressonância Magnética , Compostos Radiofarmacêuticos/química
19.
Nat Protoc ; 15(3): 1132-1157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005983

RESUMO

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.


Assuntos
Técnicas de Química Analítica/métodos , Espectrometria de Massas/métodos , Proteínas/química , Tampões (Química) , Cromatografia em Gel
20.
BMC Complement Med Ther ; 20(1): 5, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-32020890

RESUMO

BACKGROUND: Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line. METHODS: NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools. RESULTS: Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 µg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range. CONCLUSION: Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nigella sativa/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/farmacologia , Cromatografia em Gel , Humanos , Células MCF-7 , Espectrometria de Massas , Paquistão , Sementes/química
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