RESUMEN
Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO2 ) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO2 and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO2 particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO2 particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.
Asunto(s)
Fosfopéptidos , Proteómica , Cromatografía de Afinidad/métodos , Espectrometría de Masas , Fosfopéptidos/análisis , Fosforilación , Proteómica/métodos , Titanio/químicaRESUMEN
Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.
Asunto(s)
Histidina Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Quinonas/farmacología , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Histidina Quinasa/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.
Asunto(s)
Ácido Aspártico/análisis , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Colorantes Fluorescentes/análisis , Histidina/análisis , Complejos Multienzimáticos/metabolismo , Transducción de Señal/fisiología , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Colorantes Fluorescentes/metabolismo , Histidina/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/fisiología , Transactivadores/metabolismoRESUMEN
Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-µL micropipette tip containing 10 µL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 µg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
Asunto(s)
Fosfopéptidos/química , Piridinas/química , Cromatografía de Afinidad/métodos , Células HEK293 , Humanos , Fosfopéptidos/aislamiento & purificación , Fosfopéptidos/metabolismo , Fosforilación , Sensibilidad y Especificidad , Sefarosa/química , Espectrometría de Masas en Tándem/métodosRESUMEN
We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos-tag SDS-PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos-tag gels, the separation was not due to phosphorylation. The N-terminal 47-61 region of ClpX was responsible for producing multiple phosphorylation-independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up-shifted bands. By systematic Ala-scanning mutation analysis in the N-47-61 region, we concluded that the Glu-51 or Glu-54 residue was responsible for the appearance of exaggerated mobility-shifting bands. Histone H2A showed a much slower migration in Phos-tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu-62 or Glu-65 residue caused the retarded migration. In addition, Phos-tag SDS-PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos-tag SDS-PAGE is induced by interactions between the Phos-tag molecule and the carboxylate group of a specific Glu residue on the target.
Asunto(s)
Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Ácido Glutámico/química , Piridinas/farmacología , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas , Sitios de Unión , Endopeptidasa Clp , Proteínas de Escherichia coli , Histonas/análisis , Humanos , Chaperonas Moleculares , Peso Molecular , FosforilaciónRESUMEN
We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.
Asunto(s)
Fosfatasa Alcalina/análisis , Colorantes , Colorantes Fluorescentes , Piridinas , RodaminasRESUMEN
Stent perforation of a coronary artery during percutaneous coronary intervention (PCI) is a rare but life-threatening complication. A 70-year-old man was admitted to our hospital for management of acute coronary syndrome. PCI was attempted for the left circumflex artery(LCX);however, catheterization was complicated by perforation of the LCX by the stent. He developed cardiac tamponade and was resuscitated using percutaneous cardiopulmonary support. Emergency surgery( ligation of the LCX and aorto-coronary bypass grafting for the distal LCX) was performed. His postoperative course was uneventful, and the patient was discharged on the 20th postoperative day. Collaboration between cardiologists and surgeons is essential to salvage such a critical patient.
Asunto(s)
Enfermedad de la Arteria Coronaria/cirugía , Vasos Coronarios/lesiones , Intervención Coronaria Percutánea , Anciano , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Humanos , Masculino , Resultado del TratamientoRESUMEN
MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas , Proteómica/métodos , Aminoácidos/análisis , Aminoácidos/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Células HEK293 , Células HeLa , Humanos , MAP Quinasa Quinasa 1/química , FosforilaciónRESUMEN
This review article describes analytical techniques based on the phosphate-binding tag molecule "Phos-tag", which is an alkoxide-bridged dinuclear metal complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate, for studying the protein phosphorylome. The dinuclear zinc(II) complex forms a stable 1:1 complex with a phosphate monoester dianion in an aqueous solution under conditions of neutral pH. By using a series of functional Phos-tag derivatives, our group has developed novel techniques that are useful in studies on kinomics and phosphoproteomics. Among the derivatives, a series of biotinylated Phos-tag derivatives have been used as molecular tools in applications such as Western blotting for comprehensive detection of phosphorylated proteins and in highly sensitive peptide microarray-based techniques for the detection of kinase activities in biological samples. The review also gives an outline of phosphate affinity electrophoresis, in which immobilized Phos-tag molecules in a general polyacrylamide gel are used to separate proteins and detect differences in their phosphorylation status. This technique permits quantitative analyses of multiple phosphorylation statuses of individual cellular proteins and their time-dependent changes. Conventional mass spectrometry-based shotgun techniques used in phosphoproteomics detect the phosphorylation modification of proteins in peptide fragments, whereas the Phos-tag electrophoresis technique permits the direct analysis of the phosphorylation status of full-length proteins. The technique therefore provides a greater understanding of the detailed properties of particular proteins involved in specific physiological and pathological events. This article is part of a Special Issue entitled: Medical Proteomics.
Asunto(s)
Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Piridinas/química , Animales , Humanos , Fosfoproteínas/química , Proteoma/químicaRESUMEN
The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.
Asunto(s)
Western Blotting/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Fosfoproteínas/análisis , Piridinas/química , Células HeLa , Humanos , Fosfoproteínas/aislamiento & purificación , FosforilaciónRESUMEN
Adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos-tag affinity electrophoresis. The Phos-tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two-component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent-denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P-N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos-tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Proteínas Quinasas/metabolismo , Piridinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bacterias/enzimología , Bovinos , Electroforesis/métodos , Concentración de Iones de Hidrógeno , Fosforilación , Proteínas Recombinantes/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.
Asunto(s)
Cilióforos/química , Cilióforos/crecimiento & desarrollo , Regulación de la Expresión Génica , Proteínas Protozoarias/análisis , Proteínas Protozoarias/aislamiento & purificación , Esporas Protozoarias/química , Esporas Protozoarias/crecimiento & desarrollo , Actinas/análisis , Actinas/química , Actinas/aislamiento & purificación , Animales , Chaperonina 60/análisis , Chaperonina 60/química , Chaperonina 60/aislamiento & purificación , Cromatografía Liquida , Cilióforos/genética , Electroforesis en Gel Bidimensional , Peso Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Protozoarias/química , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esporas Protozoarias/genética , Espectrometría de Masas en TándemRESUMEN
A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.
Asunto(s)
Glutatión/aislamiento & purificación , Compuestos Heterocíclicos/química , Magnetismo/métodos , Zinc/química , Ciclamas , Glutatión/química , Fenómenos Magnéticos , Magnetismo/instrumentación , Péptidos/química , Péptidos/aislamiento & purificaciónRESUMEN
A 15-year-old girl had experienced hip pain at 11 years of age. At 15 years of age, the patient complained of persistent generalised pain. Her rheumatoid factor and serum matrix metalloproteinase-3 levels were below standard values; there were no inflammatory responses, and the human leukocyte antigen test was negative for B27 and positive for B52 and B62. The bath ankylosing spondylitis disease activity index (BASDAI) value was 8.0 at the time of induction and 3.1 at 6 months after the introduction of adalimumab (at a dose of 40 mg). The BASDAI value improved with an increase in the dose of adalimumab to 80 mg at 8 months after the initial introduction of adalimumab (at 40 mg), although it remained at 4.8 at 16 months after the dose increase. The BASDAI value was 2.6 at 6 months, 2.7 at 1 year, and 1.8 at 1.5 years after the introduction of infliximab, indicating that the patient had progressed well without any adverse events. Based on this case, juvenile ankylosing spondylitis is a differential diagnosis for low back pain and generalised pain since childhood. Tumour necrosis factor (TNF) inhibitors were promptly introduced in this case, although it took 4 years from the initial presentation. TNF inhibitors were effective in treating juvenile ankylosing spondylitis in the present case without any adverse events. This case is notable because juvenile onset ankylosing spondylitis is one of the reasons for severe lumbago since childhood and because TNF inhibitors were administered promptly after diagnosis.
Asunto(s)
Adalimumab , Antirreumáticos , Espondilitis Anquilosante , Factor de Necrosis Tumoral alfa , Humanos , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/diagnóstico , Femenino , Adolescente , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adalimumab/uso terapéutico , Adalimumab/administración & dosificación , Resultado del Tratamiento , Antirreumáticos/uso terapéutico , Antirreumáticos/administración & dosificación , Infliximab/uso terapéutico , Infliximab/administración & dosificación , Índice de Severidad de la EnfermedadRESUMEN
We describe two procedures for performing sandwich assays of phosphorylation of protein multiplexes by using several antibodies and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer. The first procedure is based on an antibody microarray technique with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. The second is based on the Bio-Plex suspension array technique with a flow-based microplate fluorescence reader system. By using this procedure, we demonstrated the quantitative detection of the entire level of phosphorylation in a target protein involved in intracellular signaling.
Asunto(s)
Anticuerpos , Análisis por Matrices de Proteínas/métodos , Proteínas/química , Piridinas , Biotina/química , Luminiscencia , Fosfoproteínas/metabolismo , Sensibilidad y EspecificidadRESUMEN
Malaysia is one of the top exporters of palm oil, and although currently facing fierce resistance towards palm oil imports in some parts of the globe, one of the ways to utilize this commodity is by increasing palm biodiesel content in local commercial diesel. However, due to the oxygen-rich nature of biodiesel, its utilization suffers from increased nitrogen oxides (NOx) emission compared to conventional diesel. To mitigate this issue and improve diesel engine performance and emissions using biodiesel-diesel blends, this study attempted to investigate implementation of a real-time non-surfactant emulsion fuel supply system (RTES) which produces water-in-diesel emulsion as fuel without surfactants. NOx reducing capability of water-in-diesel produced by RTES has been well documented. Therefore, in this study, 30% biodiesel-diesel (B30) was used as the base fuel while B30-derived emulsions consisting of 10 wt%, 15 wt% and 20 wt% water content were supplied into a 100 kVA, 5.9-L common rail turbocharged diesel engine electric generator. Fuel consumption and exhaust emissions were measured and compared with commercially available Malaysian low grade diesel fuel (D2M). Evidence suggested that emulsified B30 biodiesel-diesel produced by RTES was able to increase brake thermal efficiency (BTE) up to a maximum of 36% and reduce brake specific fuel consumption (BSFC) up to 8.70%. Furthermore, B30 biodiesel-diesel emulsions produced significantly less NOx, carbon monoxide and smoke at high engine load. In conclusion, B30 biodiesel-diesel emulsions can be readily utilized in current diesel engines without compromising on performance and emissions.
Asunto(s)
Surfactantes Pulmonares , Tensoactivos , Emulsiones , Biocombustibles , Aceite de Palma , LipoproteínasRESUMEN
We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) in Zn(2+)-Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10-200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris-HCl and Tris-acetic acid (Tris-AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10-200 kDa cellular proteins, the Bis-Tris-HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn(2+)-Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris-AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida/métodos , Fosfoproteínas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Supresoras de Tumor/química , Marcadores de Afinidad/química , Proteínas de la Ataxia Telangiectasia Mutada , Tampones (Química) , Ensayo de Cambio de Movilidad Electroforética/métodos , Células HeLa , Humanos , Ácido Clorhídrico/química , Concentración de Iones de Hidrógeno , Toxinas Marinas , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/química , Peso Molecular , Oxazoles/farmacología , Fosforilación , Vimentina/química , Zinc/químicaRESUMEN
We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.
Asunto(s)
Biotina/química , Fosfoproteínas/análisis , Proteómica/métodos , Piridinas/química , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Western Blotting , Bovinos , Línea Celular , Peroxidasa de Rábano Silvestre/química , Humanos , Indicadores y Reactivos/química , Mediciones Luminiscentes , Fosfopéptidos/análisis , Fosfopéptidos/química , Fosfoproteínas/química , Fosforilación , Análisis por Matrices de Proteínas , Sensibilidad y EspecificidadRESUMEN
Ovalbumin (OVA) derived from egg white contains two residues that can be phosphorylated: Ser-68 and Ser-344. Native polyacrylamide gel electrophoresis(PAGE) shows the presence of three distinct migration bands corresponding to phosphorylation states with two, one, or no phosphate groups, respectively. Phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Zn(2+)-Phos-tag SDS-PAGE), on the other hand, showed the presence of four distinct phosphorylated states in intact OVA. In addition to the diphosphorylated and nonphosphorylated forms, two distinct species, one with a phosphate group at Ser-68 and one with a phosphate group at Ser-344, were separately visualized. The content of the OVA monophosphorylated at Ser-68 was greater than that of OVA monophosphorylated at Ser-344. Zn(2+)-Phos-tag SDS-PAGE is therefore a useful method for the quantitative analysis of the detailed phosphorylation status of food proteins.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Ovalbúmina/química , Fosfoproteínas/química , Piridinas/química , Serina/química , Secuencia de Aminoácidos , Animales , Pollos , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Datos de Secuencia Molecular , FosforilaciónRESUMEN
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.