Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
1.
Artículo en Inglés | MEDLINE | ID: mdl-38718698

RESUMEN

Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel "collectors" that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography - mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.


Asunto(s)
Pruebas Respiratorias , Límite de Detección , Detección de Abuso de Sustancias , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Detección de Abuso de Sustancias/métodos , Pruebas Respiratorias/métodos , Cromatografía Liquida/métodos , Reproducibilidad de los Resultados , Metadona/análisis , Metadona/orina , Modelos Lineales , Masculino , Femenino , Adulto , Drogas Ilícitas/análisis , Drogas Ilícitas/orina , Cromatografía Líquida con Espectrometría de Masas
2.
Sci Adv ; 8(12): eabm0220, 2022 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-35333580

RESUMEN

Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the Beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 µg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.


Asunto(s)
COVID-19 , Camélidos del Nuevo Mundo , Anticuerpos de Dominio Único , Animales , Anticuerpos Monoclonales/química , Anticuerpos Antivirales , Camélidos del Nuevo Mundo/metabolismo , Humanos , Glicoproteínas de Membrana , Pruebas de Neutralización , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/metabolismo
3.
Molecules ; 26(21)2021 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-34771115

RESUMEN

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins mostly as a result of spontaneous deamidation of asparaginyl residues. An association has been found between isoAsp in human serum albumin (HSA) and Alzheimer's disease (AD). Here we report on a novel monoclonal antibody (mAb) 1A3 with excellent specificity to isoAsp in the functionally important domain of HSA. Based on 1A3 mAb, an indirect enzyme-linked immunosorbent assay (ELISA) was developed, and the isoAsp occupancy in 100 healthy plasma samples was quantified for the first time, providing the average value of (0.74 ± 0.13)%. These results suggest potential of isoAsp measurements for supplementary AD diagnostics as well as for assessing the freshness of stored donor blood and its suitability for transfusion.


Asunto(s)
Inmunoensayo/métodos , Ácido Isoaspártico/química , Albúmina Sérica Humana/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Humanos , Ácido Isoaspártico/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Sensibilidad y Especificidad , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/inmunología , Espectrometría de Masas en Tándem
4.
Structure ; 28(9): 1035-1050.e8, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32668197

RESUMEN

The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT. These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.


Asunto(s)
Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Péptidos/metabolismo , Microscopía por Crioelectrón , Humanos , Proteína Huntingtina/genética , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Espectrometría de Masas , Modelos Moleculares , Mutación , Péptidos/química , Fosforilación , Dominios Proteicos , Dispersión del Ángulo Pequeño , Serina/metabolismo , Difracción de Rayos X
5.
Science ; 362(6416): 834-839, 2018 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-30442810

RESUMEN

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Bencimidazoles/farmacología , ADN Glicosilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Expresión Génica/efectos de los fármacos , Inflamación/tratamiento farmacológico , Piperidinas/farmacología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Bencimidazoles/uso terapéutico , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Técnicas de Inactivación de Genes , Guanina/análogos & derivados , Guanina/antagonistas & inhibidores , Guanina/metabolismo , Células HEK293 , Humanos , Inflamación/genética , Células Jurkat , Ratones , Ratones Mutantes , FN-kappa B/genética , FN-kappa B/metabolismo , Piperidinas/uso terapéutico , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/farmacología
6.
J Pharm Biomed Anal ; 160: 80-88, 2018 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-30086509

RESUMEN

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.


Asunto(s)
Desarrollo de Medicamentos/métodos , Fluorometría/métodos , Péptidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Sitios de Unión , Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Luz , Péptidos/química , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/aislamiento & purificación , Dispersión de Radiación , Temperatura
7.
Amino Acids ; 48(12): 2809-2820, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27573935

RESUMEN

Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.


Asunto(s)
Amidas/inmunología , Cistatina C/inmunología , Mapeo Epitopo , Amidas/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Cistatina C/antagonistas & inhibidores , Cistatina C/química , Medición de Intercambio de Deuterio , Humanos , Ratones , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/inmunología
8.
Anal Chem ; 87(23): 11840-6, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26531800

RESUMEN

Prefractionation of proteins is often employed to improve analysis specificity in proteomics. Prefractionation based on the isoelectric point (pI) is particularly attractive because pI is a well-defined parameter and it is orthogonal to hydrophobicity on which reversed-phase chromatography is based. However, direct capillary electrophoresis of blood proteins is challenging due to its high content of salts and charged small molecules. Here, we couple an online desalinator device to our multijunction capillary isoelectric focusing (MJ-CIEF) instrument and perform direct isoelectric separation of human blood plasma. In a proof-of-principle experiment, pooled samples of patients with progressive mild cognitive impairment and corresponding healthy controls were investigated. Injection of 3 µL of plasma containing over 100 µg of proteins into the desalinator was followed by pI fractionation with MJ-CIEF in less than 1 h. Shotgun proteomics of 12 collected fractions from each of the 5 replicates of pooled samples resulted in the identification and accurate quantification (median CV between the replicates is <4%) of nearly 365 protein groups from 4030 unique peptides (with <1% FDR for both peptides and proteins). The obtained results include several proteins previously reported as AD markers. The isoelectric point of each quantified protein was calculated using a set of 7 synthetic peptides spiked into the samples. Several proteins with a significant pI shift between their isoforms in the patient and control samples were identified. The presented method is straightforward, robust, and scalable; therefore, it can be used in both biological and clinical applications.


Asunto(s)
Proteínas Sanguíneas/análisis , Disfunción Cognitiva/sangre , Internet , Focalización Isoeléctrica , Biomarcadores/análisis , Tampones (Química) , Fraccionamiento Químico , Electroforesis Capilar , Humanos
9.
J Proteome Res ; 14(10): 4179-93, 2015 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-26293246

RESUMEN

Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.


Asunto(s)
Marcaje Isotópico/métodos , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Proteómica/métodos , Proteínas Proto-Oncogénicas B-raf/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medición de Intercambio de Deuterio , Humanos , Indoles/química , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Espectrometría de Masas/métodos , Simulación de Dinámica Molecular , Mutación , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/farmacología , Péptidos/análisis , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolisis , Proteómica/instrumentación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sorafenib , Sulfonamidas/química , Sulfonamidas/farmacología , Tripsina/química , Vemurafenib
10.
PLoS One ; 10(7): e0134293, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26225432

RESUMEN

The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related γ-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb)2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.


Asunto(s)
Pseudomonas aeruginosa/enzimología , Ribonucleótido Reductasas/metabolismo , Unión Proteica
11.
Anal Chem ; 86(12): 5728-32, 2014 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-24824042

RESUMEN

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.


Asunto(s)
Focalización Isoeléctrica/instrumentación , Membranas Artificiales , Proteómica , Algoritmos , Concentración de Iones de Hidrógeno
12.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 12): 2563-79, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24311597

RESUMEN

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.


Asunto(s)
Erizos/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Mapas de Interacción de Proteínas , Transducción de Señal , Proteína con Dedos de Zinc GLI1
13.
Mol Cell Proteomics ; 12(11): 3330-8, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23878402

RESUMEN

Multiparameter optimization of an LC-MS/MS shotgun proteomics experiment was performed without any hardware or software modification of the commercial instrument. Under the optimized experimental conditions, with a 50-cm-long separation column and a 4-h LC-MS run (including a 3-h optimized gradient), 4,825 protein groups and 37,550 peptides were identified in a single run and 5,354 protein groups and 56,390 peptides in a triplicate analysis of the A375 human cell line, for approximately 50% coverage of the expressed proteome. The major steps enabling such performance included optimization of the cell lysis and protein extraction, digestion of even insoluble cell debris, tailoring the LC gradient profile, and choosing the optimal dynamic exclusion window in data-dependent MS/MS, as well as the optimal m/z scan window.


Asunto(s)
Proteoma/análisis , Proteómica/métodos , Tampones (Química) , Línea Celular , Cromatografía Liquida/métodos , Humanos , Proteoma/aislamiento & purificación , Proteómica/instrumentación , Proteómica/estadística & datos numéricos , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodos
14.
Anal Chem ; 84(15): 6856-62, 2012 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-22779778

RESUMEN

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.


Asunto(s)
Focalización Isoeléctrica/métodos , Péptidos/aislamiento & purificación , Hidróxido de Amonio , Tampones (Química) , Electroforesis Capilar/instrumentación , Hidróxidos/química , Iones/química , Focalización Isoeléctrica/instrumentación , Punto Isoeléctrico , Soluciones/química
15.
FEBS J ; 278(20): 3815-21, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21668648

RESUMEN

The study of protein structure and function has evolved to become a leading discipline in the biophysical sciences. Although it is not yet possible to determine 3D protein structures from MS data alone, multiple MS-based techniques can be combined to obtain structural and functional data that are complementary to classical protein structure information obtained from NMR or X-ray crystallography. Monitoring gas-phase interactions of noncovalent complexes yields information on binding constants, complex stability, and the nature of interactions. Ion mobility MS and chemical crosslinking strategies can be applied to probe the architecture of macromolecular assemblies and protein-ligand complexes. MS analysis of hydrogen-deuterium exchange can be used to determine the localization of secondary structure elements, binding sites and conformational dynamics of proteins in solution. This minireview focuses first on new strategies that combine these techniques to gain insights into protein structure and function. Using one such strategy, we then demonstrate how a novel hydrogen-deuterium exchange microfluidics tool can be used online with an ESI mass spectrometer to monitor regional accessibility in a peptide, as exemplified with amyloid-ß peptide 1-40.


Asunto(s)
Medición de Intercambio de Deuterio , Espectrometría de Masas , Microfluídica , Proteínas/química , Proteínas/metabolismo , Animales , Deuterio/química , Humanos , Hidrógeno/química , Modelos Moleculares , Relación Estructura-Actividad
16.
Mol Cell Proteomics ; 10(9): M110.006510, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21610101

RESUMEN

A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.


Asunto(s)
Medición de Intercambio de Deuterio/métodos , Deuterio/metabolismo , Hidrógeno/metabolismo , Espectrometría de Masas/métodos , Péptidos , Proteínas , Proteómica/métodos , Anticuerpos/metabolismo , Automatización de Laboratorios , Óxido de Deuterio/metabolismo , Cinética , Péptidos/análisis , Péptidos/química , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Desplegamiento Proteico , Proteínas/análisis , Proteínas/química
17.
Anal Bioanal Chem ; 399(1): 191-5, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20717653

RESUMEN

Downscaled analytical tools for sample preparation have offered benefits such as higher throughput, easier automation and lower sample/reagent consumption. Microfluidic electrocapture, which is a newly developed sample preparation/manipulation system, uses an electric field to trap and separate charged species without using any solid sorbent. The feasibility of using microfluidic electrocapture is reported for separation, clean-up, concentration, microreactions and complexation studies of proteins, peptides and other biologically important biomolecules. The instrumentation and applications of microfluidic electrocapture are reviewed and an overview is provided of future perspectives offered by the current and envisaged platforms.


Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/tendencias , Animales , Diseño de Equipo , Humanos , Técnicas Analíticas Microfluídicas/métodos , Péptidos/análisis , Proteínas/análisis
18.
J Mol Biol ; 404(2): 328-36, 2010 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-20887730

RESUMEN

Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.


Asunto(s)
Fibroínas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Deuterio , Dimerización , Fibroínas/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Concentración Osmolar , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masa por Ionización de Electrospray , Arañas/química , Arañas/genética , Electricidad Estática , Ultracentrifugación
19.
Biochem Biophys Res Commun ; 391(3): 1561-6, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20036640

RESUMEN

Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-muM). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.


Asunto(s)
Péptido C/química , Insulina/química , Benzotiazoles , Colorantes Fluorescentes/química , Humanos , Estabilidad Proteica , Espectrometría de Masa por Ionización de Electrospray , Tiazoles/química
20.
Anal Chem ; 80(18): 7116-20, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18693749

RESUMEN

To isolate membrane-associated proteins, which play diverse structural, catalytic, and regulatory roles in cells, they are often initially solubilized in detergents. Although detergents are essential for purifying membrane proteins, they tend to interfere strongly with subsequent analyses. A microfluidic method is presented here that surmounts this problem, allowing well-resolved mass spectra of test membrane-associated polypeptides, and their complexes with ions and detergents, to be acquired. As a front-end module it allows access to other advanced mass spectrometric strategies to be utilized toward defining biomolecular interactions. This opens up a new avenue for studying complexation and analysis of membrane proteins of general importance.


Asunto(s)
Espectrometría de Masas/métodos , Proteínas de la Membrana/aislamiento & purificación , Técnicas Analíticas Microfluídicas/métodos , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Detergentes/química , Detergentes/aislamiento & purificación , Glucósidos/química , Gramicidina/química , Gramicidina/aislamiento & purificación , Proteínas de la Membrana/química , Péptidos/química , Solubilidad
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA