RESUMO
Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.
Assuntos
Neoplasias Pancreáticas , Peptídeo Hidrolases , Ácidos Fosforosos , Humanos , Peptídeo Hidrolases/metabolismo , Proteínas , Biomarcadores , Hormônios PancreáticosRESUMO
The two ZnII atoms in the crystal structure of the title complex, [Zn(NO3)2(C10H24N4)]·CH3OH, have a distorted octa-hedral coordination sphere, defined by 1,4,8,11-tetra-aza-cyclo-tetra-decane (cyclam) N atoms in the equatorial plane and nitrate O atoms in the axial sites. The conformation of the cyclam is trans-III (R, R, S, S), which is typical for metal-cyclam complexes. Nitrate anions are involved in intra- and inter-molecular hydrogen bonding with the N-H groups of the ZnII-cyclam unit. Together with the methanol solvent mol-ecule, the hydrogen-bonding network connects the ZnII-cyclam units into ribbons running parallel to the a axis.
RESUMO
In a bacterial two-component system (TCS), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator (RR). The His- and Asp-bound phosphate groups are extremely unstable under acidic conditions easily to be hydrolyzed within a few hours. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation states in the TCS. Here, we describe that Phos-tag technique is suitable for the quantitative analysis of His- and Asp-phosphorylated proteins. The dynamics of the His-Asp phosphorelay of recombinant TCS derived from Escherichia coli, was examined by Phos-tag SDS-PAGE or Phos-tag fluorescent dye gel staining. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of HK and RR in the presence of ATP or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from HK to RR in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag fluorescent dye gel staining. Consequently, Phos-tag technique provides a simple and convenient approach for screening of HK inhibitors that have potential as new antimicrobial agents. SIGNIFICANCE: Bacterial cells have unique phosphotransfer signaling mechanisms known as two-component systems (TCSs) that permit the organism to sense and respond to various environmental conditions. Each system consists of a histidine kinase (HK) and a response regulator (RR). A typical HK contains an invariant His residue that is autophosphorylated in an ATP-dependent manner. A typical RR has a conserved Asp residue that can acquire a phosphoryl group from its cognate HK. In general, TCS has this type of a His-Asp phosphorelay scheme. Because TCS is also involved in the virulence of pathogens, it is potential targets for novel antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in the bacterial TCS. We believe that our Phos-tag technique provides a simple and convenient approach for drug discovery targeting the bacterial TCS.
Assuntos
Fosfoproteínas , Piridinas , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Histidina Quinase , Fosfoproteínas/análise , FosforilaçãoRESUMO
Phos-tag is a functional molecule that selectively captures a phosphate monoester dianion in neutral aqueous solutions. The affinity of Phos-tag for phosphate monoester dianions is more than 10,000 times greater than that for other anions present in living organisms, such as carboxylic acid anions. We have developed and applied useful techniques for phosphoproteomics based on Phos-tag. This review describes the history of Phos-tag development and outlines three main technologies that have been put to practical use. The first is a technique to separate and concentrate phosphopeptides and phosphoproteins using a Phos-tag derivative with a hydrophilic chromatography carrier (Phos-tag polymer beads). The second is a technology to detect phosphopeptides and phosphoproteins on various arrays using Phos-tag biotin. The third is a technique to separate and detect phosphoproteins by electrophoresis using Phos-tag acrylamide. We hope that these three technologies will make a significant contribution to phosphoproteomics and, ultimately, to life science research. SIGNIFICANCE: The authors found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions is suitable for the access of a phosphomonoester dianion (R-OPO32-) as a bridging ligand. A dinuclear zinc(II) complex (Zn2+-Phos-tag) strongly binds to a p-nitrophenyl phosphate dianion (Kd = 2.5 × 10-8 M) at a neutral pH. The anion selectivity indexes against SO42-, CH3COO-, Cl-, and the bisphenyl phosphate monoanion at 25 °C are 5.2 × 103, 1.6 × 104, 8.0 × 105, and > 2 × 106, respectively. We have been involved in developing technologies by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this review article, the authors introduce the impact of Phos-tag-based technological advances for phosphoproteomics.
Assuntos
Fosfopeptídeos , Fosfoproteínas , Cromatografia de Afinidade/métodos , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosforilação , Piridinas , TecnologiaRESUMO
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.
Assuntos
Fosfopeptídeos , Proteômica , Cromatografia de Afinidade/métodos , Espectrometria de Massas , Fosfopeptídeos/análise , Fosforilação , Proteômica/métodos , Titânio/químicaRESUMO
The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.
Assuntos
Sistema Livre de Células/enzimologia , Regulação da Expressão Gênica/genética , Fosforilação/genética , Quinases da Família src/genética , Animais , Escherichia coli/enzimologia , Células Germinativas/enzimologia , Células HEK293 , Humanos , Insetos/enzimologia , Coelhos , Reticulócitos/enzimologia , Triticum/enzimologia , Quinases da Família src/metabolismoRESUMO
ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure-activity relationships.
Assuntos
Proteínas de Escherichia coli , Leucemia Mielogênica Crônica BCR-ABL Positiva , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Mutação , Fosforilação , PirimidinasRESUMO
We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/análise , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/análise , Escherichia coli/metabolismo , Complexos Multienzimáticos/análise , Fosfoproteínas/análise , Proteômica , Piridinas/química , Transativadores/análise , Ácido Aspártico , Corantes Fluorescentes , Histidina , FosforilaçãoRESUMO
The cationic ZnII part of aqua-(1,4,7,10-tetra-aza-cyclo-dodeca-ne)zinc(II) bis-(perchlorate), [Zn(C8H20N4)(H2O)](ClO4)2, exhibits a slightly distorted square-pyramidal coordination environment with a water mol-ecule in the apical position. In the crystal, the macrocyclic ring alternates between two conformations with equal occupancies. Two of the three perchlorate anions are situated about a twofold rotation axis, and one of them shows disorder of the O atoms with occupancies of 0.62â (7) and 0.38â (7). In the crystal, the complexes are connected by inter-molecular hydrogen bonding via the perchlorate anions.
RESUMO
We describe here a standard protocol for determining the phosphorylation status of protein multiplexes using antibody arrays and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer (Phos-tag Biotin). The procedure is based on an antibody microarray technique used in conjunction with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. By using this procedure, we have demonstrated the quantitative detection of the entire phosphorylation status of a target protein involved in intracellular signaling.
Assuntos
Fosfoproteínas/análise , Análise Serial de Proteínas/métodos , Animais , Anticorpos/imunologia , Biotinilação , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Fosfoproteínas/imunologia , PolietilenoglicóisRESUMO
Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.
Assuntos
Caseína Quinase I/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Células HEK293/metabolismo , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Fosforilação , SerinaRESUMO
Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-affinity micropipette tip (Phos-tag tip). All steps for the phosphate-affinity separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC-MS). Thus, a combination of the Phos-tag tip method and IP-RPLC-MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.
RESUMO
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.
Assuntos
Histidina Quinase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinonas/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Histidina Quinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin ß1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.
Assuntos
Forminas/metabolismo , Piridinas/química , Serina/química , Animais , Células COS , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Forminas/química , Forminas/genética , Células HEK293 , Humanos , Mutação , FosforilaçãoRESUMO
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.
Assuntos
Ácido Aspártico/análise , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/análise , Histidina/análise , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/fisiologia , Ácido Aspártico/metabolismo , Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/metabolismo , Histidina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Transativadores/metabolismoRESUMO
The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.
Assuntos
MAP Quinase Quinase 1/genética , Neoplasias/genética , Linhagem Celular , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/enzimologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Anticorpos Monoclonais , Eletroforese em Gel de Poliacrilamida/métodos , Células HL-60 , Humanos , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteômica/métodosRESUMO
Thiolate coordination to zinc(ii) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)-zinc(ii) complex (TAMRA-ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA-Cys) with TAMRA-ZnL (each at 2.5 µM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(ii) complex]/[uncomplexed TAMRA-ZnL][uncomplexed TAMRA-Cys], M-1) was determined to be 106.7 M-1 from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-l-cysteine, l-glutathione, and N-acetyl-l-cysteinamide, were evaluated to have similar values of about 104 M-1. As a result of the ligand affinities to TAMRA-ZnL, nonlabeled zinc(ii)-cyclen induced remarkable stabilization of the reduced form of l-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.
RESUMO
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.
Assuntos
Fosfatase Alcalina/análise , Corantes , Corantes Fluorescentes , Piridinas , RodaminasRESUMO
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.