RESUMEN
Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.
Asunto(s)
Biomarcadores , Electroforesis Capilar , Polisacáridos , Electroforesis Capilar/métodos , Biomarcadores/análisis , Polisacáridos/análisis , Humanos , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Microfluídica/métodos , Microfluídica/instrumentación , GlicosilaciónRESUMEN
We present in this study a new microfluidic droplet platform, named Lab-in-Droplet, for multistep glycoprotein sample treatment. Several operations are required for the sample treatment of a given glycoprotein to profile its N-glycans. In our case, all preparation steps for the analysis of N-glycans from glycoproteins could be realized in an automatic manner and without cross contamination. This could be achieved through several features that are not met in previous droplet setups, notably full automation, droplet sensing and heating. The magnetic tweezer technology was employed to manipulate (capture and release) coated magnetic beads used as analyte cargos over droplets. Droplets ranging from 1 to 10 µL play the role of confined microreactors, allowing to realize several steps that involve advanced functions such as heating and mixing with organic solvents. A complex sample treatment protocol that has been feasible so far only in batchwise mode can now be converted into a novel microfluidic version. With this Lab-in-Droplet, we can enzymatically release and fluorescently label N-linked oligosaccharides from Human Immuglobulin G and then off-line analyze the labeled glycans by capillary electrophoresis with laser induced fluorescent detection. We demonstrated the superiority of this Lab-in-Droplet over the conventional batchwise protocol, with 10-fold less reagent consumption, 3-fold less time, and 2-fold improvement of glycan labeling yield, without degradation of glycan separation profile obtained by capillary electrophoresis. The platform with the developed droplet protocol was applied successfully for mapping N-linked glycans released from human sera, serving for diagnostic screening of congenital disorders of glycosylation.
Asunto(s)
Trastornos Congénitos de Glicosilación , Trastornos Congénitos de Glicosilación/diagnóstico , Electroforesis Capilar/métodos , Glicoproteínas , Glicosilación , Humanos , Polisacáridos/análisisRESUMEN
In this study, we present a new approach for in-capillary fluorescent labeling of N-glycans prior to their analysis with CE coupled with laser-induced fluorescent detection. This integrated approach allows using a CE capillary as a microreactor to perform several steps required for labeling glycans with 8-aminopyrene-1,3,6 trisulfonic acid and at the same time as a separation channel for CE of fluorescently labeled glycans. This could be achieved through careful optimization of all different steps, including sequential injections of fluorescent dye and glycan plugs, mixing by transverse diffusion of laminar flow profiles, incubation in a thermostatic zone, and finally separation and detection with CE. Such a complex sample treatment protocol for glycan labeling that is feasible thus far only in batchwise mode can now be converted into an automated and integrated protocol. Our approach was applied successfully to analyze fluorescently labeled N-linked oligosaccharides released from human immunoglobulin G and rituximab, a monoclonal antibody used for cancer treatment. We demonstrated the superiority of this in-capillary approach over the conventional in-tube protocol, with fourfold less reagent consumption and full automation without remarkable degradation of the glycan separation profile obtained by capillary electrophoresis.
Asunto(s)
Colorantes Fluorescentes , Pirenos , Anticuerpos Monoclonales , Glicoproteínas , Humanos , Inmunoglobulina G , Oligosacáridos , Polisacáridos/análisis , RituximabRESUMEN
We present in this study a novel strategy to drastically improve the detection sensitivity and peak capacity for capillary electrophoresis with laser induced fluorescent detection (CE-LIF) of glucose oligomers and released glycans. This is based on a new approach exploiting a polymer-free background electrolyte (BGE) for CE-LIF of glycans. The best performance in terms of sample stacking and suppression of electroosmotic flow (EOF) was found for a BGE composed of triethanolamine/citric acid and triethanolamine/acetic acid at elevated ionic strengths (IS up to 200 mM). Compared to the conventional protocols for CE-LIF of glucose-oligosaccharides and released glycans, our polymer-free strategy offered up to 5-fold improvement of detection sensitivity and visualization of higher degree of polymerization (DP) of glucose oligomers (18 vs 15). To further improve the detection sensitivity, a new electrokinetic preconcentration strategy via large volume sample stacking with electroosmotic modulation without having recourse to neutrally coated capillaries is proposed, offering a 200-fold signal enhancement. This approach is based on variation of the buffer's IS, rather than pH adjustment as in conventional methods, for EOF modulation or quasi-total reduction. This strategy allows selecting with high flexibility the best pH conditions to perform efficient preconcentration and separation. The new approach was demonstrated to be applicable for the analysis of N-linked oligosaccharides released from a model glycoprotein (Human Immunoglobulin G) and applied to map N-glycans from human serum for congenital disorders of glycosylation (CDG) diagnosis.
Asunto(s)
Electroósmosis , Electroforesis Capilar , Electrólitos , Humanos , Oligosacáridos , PolisacáridosRESUMEN
Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Astrocitos/metabolismo , Disfunción Cognitiva/metabolismo , Glucólisis , Serina/biosíntesis , Administración Oral , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Sitios de Unión , Encéfalo/patología , Encéfalo/fisiopatología , Disfunción Cognitiva/patología , Disfunción Cognitiva/fisiopatología , Metabolismo Energético/efectos de los fármacos , Femenino , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Plasticidad Neuronal/efectos de los fármacos , Fosfoglicerato-Deshidrogenasa/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/administración & dosificación , Serina/farmacología , Serina/uso terapéutico , Memoria Espacial/efectos de los fármacosRESUMEN
ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs < 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.
Asunto(s)
Apolipoproteína C-III/análisis , Artefactos , Electroforesis Capilar/métodos , Glicoproteínas/análisis , Aminoácidos/química , Tampones (Química) , Glicosilación , Humanos , Neuraminidasa/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Rayos UltravioletaRESUMEN
Therapeutic proteins can easily undergo chemical or physical changes during their manufacturing, purification, and storage. These modifications might change or reduce their biological activity. Therefore, it is important to have analytical methodologies that are able to reliably detect, characterize, and quantify degradation products in formulations. Capillary Zone Electrophoresis (CZE) is very well suited for the analysis of proteins due to its relatively easiness of implementation, separation efficiency, and resolving power. We describe here a CZE method that allows separating more than nine forms in therapeutic albumin, including oxidized, glycated, and truncated forms. This method uses a polyethylene oxide (PEO) coating and a buffer composed of HEPES and SDS at physiological pH. The method is reproducible (RSD < 0.5 and 4 % for migration times and peak areas, respectively) and allows quantitation of albumin forms in pharmaceutical preparations.
Asunto(s)
Electroforesis Capilar/métodos , Albúmina Sérica Humana/química , Productos Finales de Glicación Avanzada , Humanos , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Polietilenglicoles/química , Albúmina Sérica/aislamiento & purificación , Albúmina Sérica GlicadaRESUMEN
Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.
Asunto(s)
Antitrombina III/aislamiento & purificación , Electroforesis Capilar/métodos , Tampones (Química) , Dimerización , Composición de Medicamentos , Electroforesis Capilar/instrumentación , Polietilenglicoles , Conformación Proteica , Isoformas de Proteínas/aislamiento & purificaciónRESUMEN
With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.97), which is not significantly different from the value obtained from a higher number of measurements. Due to the relatively short time required to determine the Kd of one AT variant (2h), this method has the potential for being a low throughput screening method. The method was validated by analyzing five AT variants, whose Kd have been reported in the literature using fluorescence spectroscopy. Finally, the method was applied to estimate the Kd of one new AT variant and one AT conformer, a latent form, that exhibits a significant loss of affinity.
Asunto(s)
Antitrombinas/química , Heparina/química , Técnicas de Cultivo de Célula/métodos , Electroforesis Capilar/métodos , Humanos , Cinética , Espectrometría de Fluorescencia/métodosRESUMEN
An in-line SPE method coupled to CE was developed for the analysis of DNA. The amino silica monolith was prepared in situ by polymerization of tetraethoxysilane and N-(ß-aminoethyl)-γ-aminopropyltriethoxysilane in ethanol aqueous solution at the inlet end of a 100 µm id fused-silica capillary, and the remaining part of the capillary was used as separation channel. The procedure for this in-line SPE-CE method was constructed on the basis of investigation on operational conditions such as the introduction mode of sieving matrix, the composition of elution solvent and the elution time. Twenty millimolar ammonium hydroxide was demonstrated to be effective for DNA desorption from the monolith, and linear poly(N-isopropylacrylamide) was used as the separation matrix. The proposed method could achieve limits of detection of 0.065-0.123 ng/mL for six DNA fragments ranging 100-2000 bp. Compared with conventional CE, preconcentration factors of over 100 times were obtained. The applicability of the in-line SPE-CE method was further demonstrated by analyzing plasmid DNA from Escherichia coli crude lysate.
Asunto(s)
ADN/aislamiento & purificación , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Extracción en Fase Sólida/instrumentación , Extracción en Fase Sólida/métodos , Hidróxido de Amonio , ADN/química , Escherichia coli , Hidróxidos/química , Microscopía Electrónica de Rastreo , Plásmidos/aislamiento & purificación , Sensibilidad y Especificidad , Dióxido de Silicio/químicaRESUMEN
We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.
Asunto(s)
Productos Biológicos/química , Electroforesis Capilar/métodos , Albúmina Sérica/análisis , Productos Biológicos/análisis , Biofarmacia , Humanos , Oxidación-Reducción , Polietilenglicoles/química , Albúmina Sérica/químicaRESUMEN
Lab-on-a-chip electrophoresis is becoming increasingly useful for protein analysis, thanks to recent developments in this field. This review is an update of the review we published at the start of 2008 [Peng, Y., Pallandre, A., Tran, N. T., Taverna, M., Electrophoresis 2008, 29, 156-177]. The superiority of polymers for the manufacture of analytical microchips has been confirmed. This trend implies several modifications to the processes previously used with glass/silicon chips and requires a better understanding of the interfacial phenomena of these materials. Significant progress in chip-based techniques for protein analysis has been made in the last 2 years. In addition to advances in traditional electrokinetic modes, counter-flow gradient focusing techniques have emerged as useful methods not only for separation, but also for the online preconcentration of samples. This review, with more than 175 references, presents recent advances and novel strategies for EOF measurement, surface treatment, sample pretreatment, detection and innovations relating to the different modes of separation.
Asunto(s)
Electroforesis Capilar/métodos , Dispositivos Laboratorio en un Chip , Procedimientos Analíticos en Microchip/métodos , Proteínas/análisis , Cromatografía Capilar Electrocinética Micelar/métodos , Electroósmosis/métodos , Miniaturización/métodosRESUMEN
This work reports the validation of a simple CZE method to be used in quality control of recombinant human glycosylated interleukin-7 (rhIL-7) batches produced in Chinese Hamster Ovary (CHO) cells. The separation buffer was a 25mM sodium borate at pH 10 containing 12mM diaminobutane (DAB) used as a dynamic coating agent of the capillary. This method allowed the separation of seven peaks ranging from low to high sialylated glycoforms. An extensive study on conditioning methods of the capillary has been conducted to yield repeatable results. Excellent RSD of EOF mobility (less than 0.6%) was obtained when conditioning included capillary equilibration under virtual analyses and storage in 0.1M NaOH overnight. Method specificity has been demonstrated to be able to discriminate different rhIL-7 glycoforms produced in CHO from formulation matrix. Linearity was demonstrated between 0.5 and 4mg/mL. LOQ was 0.5mg/mL. Repeatability (RSD<1.4 and 3.3% for t(m) and A%, respectively), intermediate precision of inter-day (RSD<2.1 and 4.5), inter-analyst (RSD<2.0 and 3.0) and inter-equipment (RSD<3.8 and 3.7 for electrophoretic mobility and A%, respectively) were all very satisfactory. Evaluation of robustness revealed that pH and DAB concentration are critical parameters in the method while slight alteration of ionic strength of electrolyte or change of capillary source did not affect the results. Finally the method was shown to provide reliable informations to address comparability studies and batch-to-batch consistency of biomanufactured rhIL-7.
Asunto(s)
Electroforesis Capilar , Interleucina-7/biosíntesis , Tecnología Farmacéutica/métodos , Animales , Tampones (Química) , Células CHO , Cricetinae , Cricetulus , Estabilidad de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Interleucina-7/normas , Variaciones Dependientes del Observador , Concentración Osmolar , Desnaturalización Proteica , Estabilidad Proteica , Control de Calidad , Proteínas Recombinantes/biosíntesis , Reproducibilidad de los ResultadosRESUMEN
The potential of a series of newly synthesized poly(N,N-dimethylacrylamide) (PDMA) grafted polyacrylamide (PAM) copolymers (P(AM-PDMA)) as a replaceable separation medium for protein analysis was studied. A comparative study with and without copolymers was performed; the separation efficiency, analysis reproducibility and protein recovery proved that the P(AM-PDMA) copolymers were efficient in suppressing the adsorption of basic proteins onto the silica capillary wall. Furthermore, the size-dependent retardation of native proteins in a representative P(AM-PDMA) copolymer was demonstrated by Ferguson analysis. The results showed that the P(AM-PDMA) copolymers combine the good coating property of PDMA and the sieving property of PAM and could be applied as a sieving matrix for the analysis of native proteins.
Asunto(s)
Acrilamidas/química , Electroforesis Capilar/métodos , Polímeros/química , Proteínas/aislamiento & purificaciónRESUMEN
In order to study kin17 protein-DNA affinity, we have developed a fast and reproducible capillary electrophoresis (CE) analysis of a strongly basic protein: kin17 protein, using a nonpermanent coating based on poly(ethylene oxide) (PEO) to avoid adsorption of kin17. The coating procedure was optimized to provide a residual and stable electroosmotic flow (EOF = 5 x 10(-5) cm(2)/V x s), exhibiting RSD of 0.3% and excellent long-term stability. Good intraday and interday reproducibility of kin17 migration times (0.8 and 0.3% relative standard deviation (RSD), respectively) enabled us to consider that the recovery percentage obtained for kin17 protein was satisfactory (79%). The potential of this PEO-based coating procedure was evaluated for affinity CE method in order to study the affinity of kin17 protein for two single-stranded DNA (ssDNA) models: polydeoxyadenylic acid and polydeoxycytidilic acid (pdA and pdC). Binding constants (1.5 x 10(7) +/- 17% and 1.7 x 10(7) + 25%M(-1)) were evaluated assuming a 1:1 affinity between kin17 and pdA or pdC, respectively.