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Two-dimensional Fourier transform ion cyclotron resonance (2D FTICR) mass spectrometry is a developing form of data-independent acquisition that allows for the simultaneous fragmentation and correlation of fragment ions to their precursors across a range of m/z values. The modern usage of 2D FTICR is performed using electrospray ionization (ESI) as the dried droplet preparation for matrix-assisted laser desorption ionization (MALDI) does not produce a consistent packet of ions over a number of scans. This work uses pneumatic spray techniques from mass spectrometry imaging to create a homogeneous surface for use with MALDI as an ionization source for 2D FTICR. A mixture of peptides and matrix was deposited onto a glass slide using an HTX pneumatic sprayer. MALDI was then used to ionize the peptide mixture for use with a standard 2D FTICR pulse sequence. The generated 2D spectrum reveals comparable structural information to spectra collected in a 1D experiment. Artifacts observed in the collected 2D MALDI spectra do not significantly differ from those expected from 2D ESI spectra.
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Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug-protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.
Assuntos
Proteínas Sanguíneas , Eletroforese Capilar , Eletroforese Capilar/métodos , Proteínas Sanguíneas/química , Interações MedicamentosasRESUMO
Antibody-based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side-effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography-mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis.
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Produtos Biológicos , Anticorpos/análise , Humanos , Imunoensaio/métodos , Ressonância de Plasmônio de SuperfícieRESUMO
High-performance affinity microcolumns were used to characterize binding by the anti-diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate-to-high affinity regions and a larger set of weaker regions with association equilibrium constants of â¼105 and 103 M-1 , respectively, at pH 7.4 and 37°C. Competition studies gave site-specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104 M-1 for normal albumin, with a decrease of 26%-30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide had association constants for normal albumin of 6.1 × 104 and 7.1 × 105 M-1 , with glycated albumin giving an increase in the association constant at this site for repaglinide of 1.6- to 1.8-fold and a decrease for nateglinide of 51%-58%.
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Albuminas , Albumina Sérica Humana , Humanos , NateglinidaRESUMO
Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.
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Cromatografia de Afinidade , Adsorção , LigantesRESUMO
Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.
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The authors would like to call the reader's attention to the following corrections in this article. In the description given for the process of preparing glycated human serum albumin under "In vitro glycation of HSA", the concentrations of D-glucose that were employed were 15 mM and 30 mM.
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A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.
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Proteínas Sanguíneas/química , Cromatografia de Afinidade/métodos , Preparações Farmacêuticas/química , Humanos , Cinética , Ligação Proteica , Albumina Sérica/químicaRESUMO
Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.
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Cromatografia de Afinidade/métodos , Sítios de Ligação , Ligação Proteica , TermodinâmicaRESUMO
Protein G can be a valuable binding agent for antibodies and immunoglobulins in methods such as immunosensors, chromatographic-based immunoassays, and immunoaffinity chromatography. This report used the method of peak decay analysis along with frontal analysis and zonal elution studies to characterize the binding, elution and regeneration properties of affinity microcolumns that contained immobilized protein G. Frontal analysis was employed with rabbit immunoglobulin G (IgG) to characterize the binding capacity of these affinity microcolumns. Zonal elution experiments looking at the retained peaks for small injections of labeled rabbit IgG were used to optimize the column regeneration conditions. Peak decay analysis was then used to look at the effects of flow rate and elution pH on the release of several types of IgG from the protein G microcolumns. This approach made it possible to obtain detailed information on the use and behavior of such columns, as could be used in future work to optimize the capture or analysis of IgG and antibodies by such devices. The same approach and tools that were used in this report could also be adapted for work with affinity columns that make use of other supports, binding agents or targets.
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Cromatografia de Afinidade , Imunoglobulina G/química , Animais , Imunoensaio , Cinética , Ligação Proteica , CoelhosRESUMO
The last few decades have witnessed the development of many high-performance separation methods that use biologically related binding agents. The combination of HPLC with these binding agents results in a technique known as high performance affinity chromatography (HPAC). This review will discuss the general principles of HPAC and related techniques, with an emphasis on their use for the analysis of biological compounds and pharmaceutical agents. Various types of binding agents for these methods will be considered, including antibodies, immunoglobulin-binding proteins, aptamers, enzymes, lectins, transport proteins, lipids, and carbohydrates. Formats that will be discussed for these methods range from the direct detection of an analyte to indirect detection based on chromatographic immunoassays, as well as schemes based on analyte extraction or depletion, post-column detection, and multi-column systems. The use of biological agents in HPLC for chiral separations will also be considered, along with the use of HPAC as a tool to screen or study biological interactions. Various examples will be presented to illustrate these approaches and their applications in fields such as biochemistry, clinical chemistry, and pharmaceutical research.
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Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Anticorpos/química , Carboidratos/química , Imunoensaio , Lipídeos/química , Preparações Farmacêuticas/análise , Proteínas/químicaRESUMO
Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides, and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions than a traditional C18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry.
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Hormônios/análise , Praguicidas/análise , Preparações Farmacêuticas/análise , Soroalbumina Bovina/química , Poluentes Químicos da Água/análise , Adsorção , Animais , Sítios de Ligação , Bovinos , Cromatografia Líquida/instrumentação , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
BACKGROUND: The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically related binding agent, are 2 methods that can be used to study these interactions. CONTENT: This review presents various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. SUMMARY: The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples.
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Química Clínica , Cromatografia de Afinidade , Preparações Farmacêuticas/análise , Cromatografia Líquida de Alta Pressão , Ensaios de Triagem em Larga Escala , Humanos , Medicina de PrecisãoRESUMO
Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological-binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose, and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined.
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Cromatografia de Afinidade , Proteínas/análise , Proteínas/química , Proteínas/isolamento & purificação , EstereoisomerismoRESUMO
The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent immobilization, adsorption, entrapment, and the synthesis or direct development of nanomaterials as part of a chromatographic support. Nanomaterials have been used in many types of LC. These applications have included the reversed-phase, normal-phase, ion-exchange, and affinity modes of LC, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. Both small and large analytes (e.g., dyes, drugs, amino acids, peptides and proteins) have been used to evaluate possible applications for these nanomaterial-based methods. The use of nanomaterials in columns, capillaries and planar chromatography has been considered as part of these efforts. Potential advantages of nanomaterials in these applications have included their good chemical and physical stabilities, the variety of interactions many nanomaterials can have with analytes, and their unique retention properties in some separation formats.
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Cromatografia Líquida/métodos , Nanoestruturas/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Carbono/química , Cromatografia Líquida/instrumentação , Corantes/química , Corantes/isolamento & purificação , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Metais/química , Óxidos/química , Peptídeos/química , Peptídeos/isolamento & purificação , Preparações Farmacêuticas/química , Preparações Farmacêuticas/isolamento & purificação , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.
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Peptídeos/química , Albumina Sérica/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , HumanosRESUMO
Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 µL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.
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Cromatografia de Afinidade/métodos , Diabetes Mellitus/sangue , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/isolamento & purificação , Diabetes Mellitus/tratamento farmacológico , HumanosRESUMO
An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) µg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.
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Cromatografia de Afinidade/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Orosomucoide/metabolismo , Preparações Farmacêuticas/metabolismo , Anti-Hipertensivos/isolamento & purificação , Anti-Hipertensivos/metabolismo , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Desenho de Equipamento , Humanos , Orosomucoide/isolamento & purificação , Preparações Farmacêuticas/isolamento & purificação , Propranolol/isolamento & purificação , Propranolol/metabolismo , Ligação Proteica , Varfarina/isolamento & purificação , Varfarina/metabolismoRESUMO
Ultrafast affinity extraction was used to study hormone-protein interactions in solution, using testosterone and its transport proteins human serum albumin (HSA) and sex hormone binding globulin (SHBG) as models. Both single column and two-dimensional systems based on HSA microcolumns were utilized to measure the free fraction of testosterone in hormone/protein mixtures at equilibrium or that were allowed to dissociate for various lengths of time. These data were used to determine the association equilibrium constants (Ka) or global affinities (nKa') and dissociation rate constants (kd) for testosterone with soluble HSA and SHBG. This method was also used to measure simultaneously the free fraction of testosterone and its equilibrium constants with both these proteins in physiological mixtures of these agents. The kd and Ka values obtained for HSA were 2.1-2.2 s(-1) and 3.2-3.5 × 10(4) M(-1) at pH 7.4 and 37 °C. The corresponding constants for SHBG were 0.053-0.058 s(-1) and 0.7-1.2 × 10(9) M(-1). All of these results gave good agreement with literature values, indicating that this approach could provide information on a wide range of rate constants and binding strengths for hormone-protein interactions in solution and at clinically relevant concentrations. The same method could be extended to alternative hormone-protein systems or other solutes and binding agents.
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Albumina Sérica/análise , Albumina Sérica/química , Globulina de Ligação a Hormônio Sexual/análise , Globulina de Ligação a Hormônio Sexual/química , Testosterona/análise , Testosterona/química , Sítios de Ligação , Cromatografia Líquida de Alta Pressão/instrumentação , Humanos , SoluçõesRESUMO
In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes.