RESUMO
Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E(â)) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E(â) for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E(â) for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(â) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists.
Assuntos
Marcação por Isótopo , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo , Alquilação , Humanos , Espectrometria de Massas , Oxirredução , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiorredoxinas/químicaRESUMO
Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.
Assuntos
Ácidos Graxos Monoinsaturados/química , Serina C-Palmitoiltransferase/antagonistas & inibidores , Cristalografia por Raios X , Cinética , Mutação , Proteínas Recombinantes/química , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/genética , Sphingomonas/enzimologia , Sphingomonas/genéticaRESUMO
The organometallic anticancer complex [(η(6)-bip)Ru(en)Cl](+) (1; bip = biphenyl, en = ethylenediamine) selectively binds to N7 of guanine bases of oligonucleotides and native DNA. However, under physiologically relevant conditions (micromolar Ru concentrations, pH 7, 22 mM NaCl, 310 K), the tripeptide glutathione (γ-L-Glu-L-Cys-Gly; GSH) is kinetically competitive with guanine (as guanosine 3',5'-cyclic monophosphate, cGMP) for coordination with complex 1, and gives rise to a ruthenium thiolato adduct. This thiolato adduct can subsequently undergo oxidation to a sulfenate intermediate, providing a facile route for the formation of a final cGMP adduct via the displacement of S-bound glutathione by G N7 (F. Y. Wang, J. J. Xu, A. Habtemariam, J. Bella and P. J. Sadler, J. Am. Chem. Soc., 2005, 127, 17734). In this work, the competition between GSH and the single-stranded 14-mer oligonucleotide 5'-TATGTACCATGTAT-3' (I) and duplex III (III = I + II, II = 5'-ATACATGGTACATA) for complex 1 and its analogue [(η(6)-tha)Ru(en)Cl](+) (2, tha = tetrahydroanthracene) under physiologically relevant conditions was investigated using conventional ESI-MS and high resolution ESI-FTICR-MS coupled to conventional HPLC and nanoscale HPLC, respectively. The results indicate that whether there was high excess of GSH or not in the reaction mixtures, the reaction of complex 1 or 2 with single-stranded oligonucleotide I always gave rise to mono-ruthenated oligonucleotide, and the reaction of complex 1 or 2 with duplex III gave rise to the mono-ruthenated duplex oligonucleotide. Furthermore, the ruthenation of duplex III by complex 1 showed no significant discrimination between the complementary strands I and II, but complex 2 appeared to bind preferentially to strand II compared to strand I as revealed by the high resolution FTICR-MS analysis. GSH is highly abundant in cells at millimolar concentrations and is well known to be involved in the deactivation of the clinical drug cisplatin and in platinum resistance. Our findings reveal a potentially contrasting role for GSH in the mechanism of action of these ruthenium anticancer complexes that may contribute to the lack of cross-resistance with platinum drugs.
Assuntos
Ligação Competitiva , DNA/metabolismo , Glutationa/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Rutênio/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sequência de Bases , DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Oligodesoxirribonucleotídeos/genéticaRESUMO
Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis.
Assuntos
Microtomia , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Condutividade Térmica , Imagem Corporal Total , Animais , Carbono/farmacologia , Diagnóstico por Imagem , Técnicas Histológicas , Temperatura Alta , Masculino , Inclusão em Parafina , Ratos , Restrição Física/métodos , Restrição Física/fisiologia , Manejo de Espécimes/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Fita Cirúrgica/estatística & dados numéricos , Imagem Corporal Total/métodos , Imagem Corporal Total/veterináriaRESUMO
Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. Mössbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.
Assuntos
Proteínas de Bactérias/química , Compostos Férricos/química , Proteínas de Ligação ao Ferro/química , Proteínas de Bactérias/genética , Sítios de Ligação , Calorimetria , Clonagem Molecular , Proteínas de Ligação ao Ferro/genética , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Neisseria gonorrhoeae , Espectroscopia de MossbauerRESUMO
Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.
Assuntos
Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Ácido Láctico/farmacologia , Acetilação , Ânions , Butiratos/farmacologia , Linhagem Celular , Meios de Cultura/química , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Ácido Láctico/análiseRESUMO
Oxidation of cysteine is now known to serve as a fundamental mechanism to control protein function or activity. Many redox-regulated proteins do not oxidize to homogeneity, resulting in a mixture of reduced and oxidized species which cannot be separated chromatographically. Here we describe a protocol for the separation of reduced and oxidized forms of the tumor suppressor protein p53. This purification method relies on the reversible labeling of thiol groups with biotin and exploitation of the ultrastrong biotin-avidin interaction. This purification procedure can be applied to other cysteine-containing proteins where enrichment of the oxidized form is required.
Assuntos
Cromatografia de Afinidade/métodos , Proteína Supressora de Tumor p53/isolamento & purificação , Proteína Supressora de Tumor p53/metabolismo , Avidina/química , Biotina/química , Cisteína/química , Humanos , Oxirredução , Compostos de Sulfidrila/químicaRESUMO
Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.
Assuntos
Análise de Fourier , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Elétrons , Humanos , Dados de Sequência Molecular , Mucoproteínas , Proteínas Oncogênicas , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas/metabolismoRESUMO
The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.
Assuntos
Cisteína/química , Espectrometria de Massas/métodos , Proteína Supressora de Tumor p53/química , Alquilantes , Sequência de Aminoácidos , Cristalografia por Raios X , Cisteína/metabolismo , Etilmaleimida , Análise de Fourier , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Desdobramento de Proteína , Temperatura , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.
Assuntos
Análise de Fourier , Histocitoquímica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Química Encefálica , Rim/química , Metabolômica , Camundongos , Fosfolipídeos/análise , Compostos de EstanhoRESUMO
We have developed an automated quench-flow microreactor which interfaces directly to an electrospray ionization (ESI) mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen to test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FTICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme intermediate using top-down fragmentation. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assigned with high confidence. Electron capture dissociation (ECD) fragmentation allowed us to locate the intermediate to a five amino acid section of the protein--which includes the known catalytic residue, Ser(195). This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms, is a potentially powerful tool for studies of enzyme catalysis.
Assuntos
Quimotripsina/metabolismo , Ciclotrons , Análise de Fourier , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Quimotripsina/química , Cinética , Dados de Sequência MolecularRESUMO
Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/química , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Catálise , Linhagem Celular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADP/metabolismo , Oxirredução , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Transporte Proteico , Compostos de Sulfidrila/química , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Bacterioferritin comigratory protein (BCP) is a bacterial thioredoxin-dependent thiol peroxidase that reduces a variety of peroxide substrates. Using high-resolution Fourier transform ion cyclotron resonance mass spectrometry coupled with top-down fragmentation techniques, we have analyzed the mechanistic details of hydrogen peroxide reduction by E. coli BCP. We show here that catalysis occurs via an atypical two-cysteine peroxiredoxin pathway. A transient sulfenic acid is initially formed on Cys-45, before resolution by the formation of an intramolecular disulfide bond between Cys-45 and Cys-50. This oxidized BCP intermediate is shown to be a substrate for reduction by thioredoxin, completing the catalytic cycle. Although we invoke Cys-50 in the catalytic cycle of Escherichia coli bacterioferritin comigratory protein (BCP), a previous study had shown that this residue was not absolutely required for peroxiredoxin activity. In order to explain these apparently conflicting phenomena, we analyzed the reaction of a C50S BCP mutant with peroxide. We show that this mutant BCP enzyme adopts a different and novel mechanistic pathway. The C50S BCP mutant reacts with peroxide to form a sulfenic acid on Cys-45, in the same manner as wild-type BCP. However, the nascent intermediate is then resolved by reaction with Cys-45 from a second BCP molecule, resulting in a dimeric intermediate containing an intermolecular disulfide bond. We further show that this novel resolving complex is a substrate for reduction by thioredoxin. The importance of our results in furthering the understanding of catalysis within BCP family is discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ferritinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biocatálise , Cromatografia de Afinidade , Grupo dos Citocromos b/química , Proteínas de Escherichia coli/química , Ferritinas/química , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Tiorredoxinas/metabolismoRESUMO
We describe a quantitative Fourier transform ion cyclotron resonance mass spectrometric (FTICR MS) analysis of the relative proportions of post-translational modification states (PTMs) of core histones in cultured cells and tissues. A novel preseparation process using a monolithic column interfaced to a 12 T FTICR MS equipped with electron capture dissociation (ECD) yields very high mass accuracy spectra, allowing direct assignment of the PTMs present in the dominant modification states of intact H4, resolving a well recognized ambiguity between trimethylation and acetylation states. By eliminating preseparation, we also obtain a highly quantitative analysis of the distribution of H4 PTMs. Rapid, extensive, and reversible effects on PTMs induced by a histone deacetylase inhibitor indicate that H4 and other core histones are accessible to modification throughout the chromatin, not just in regions of active transcription. These methods provide tools for analysis of the histone code and its role in chromatin function.
Assuntos
Ciclotrons , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Acetilação/efeitos dos fármacos , Ácidos/química , Relação Dose-Resposta a Droga , Elétrons , Células HCT116 , Inibidores de Histona Desacetilases , Histona Desacetilases/química , Histonas/isolamento & purificação , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Isótopos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Reactions of the anticancer complex [(eta(6)-bip)Ru(en)Cl](+) (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione (gamma-L-Glu-L-Cys-Gly; GSH), the abundant intracellular thiol, in aqueous solution give rise to two ruthenium cluster complexes, which could not be identified by electrospray mass spectrometry (ESI-MS) using a quadrupole mass analyzer. Here we use Fourier transform ion cyclotron mass spectrometry (nanoLC-FT-ICR MS) to identify the clusters separated by nanoscale liquid chromatography as the tetranuclear complex [{(eta(6)-bip)Ru(GSO(2))}(4)](2-) (2) and dinuclear complex [{(eta(6)-bip)Ru(GSO(2))(2)}(2)](8-) (3) containing glutathione sulfinate (GSO(2)) ligands. Use of (18)OH(2) showed that oxygen from water can readily be incorporated into the oxidized glutathione ligands. These data illustrate the power of high-resolution MS for identifying highly charged multinuclear complexes and elucidating novel reaction pathways for metallodrugs, including ligand-based redox reactions.
Assuntos
Antineoplásicos/química , Marcação por Isótopo/métodos , Nanotecnologia/métodos , Compostos Organometálicos/química , Compostos de Rutênio/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ciclotrons , Glutationa , Isótopos de Oxigênio , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.
Assuntos
Aptâmeros de Peptídeos/química , Biomarcadores/química , Análise Serial de Proteínas , Proteínas/química , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Técnicas de Química Combinatória , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Mucoproteínas , Proteínas Oncogênicas , Proteínas/isolamento & purificação , SuínosRESUMO
The high mass accuracy of FT-ICR MS combined with (15)N-labelling shows that mono- and di- platinated products from the reaction of erythrocyte superoxide dismutase with the anticancer drug cisplatin in solution retain their ammine ligands, in contrast to a recent X-ray crystallographic study.
Assuntos
Amônia/química , Cisplatino/química , Íons/química , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Ligantes , Espectrometria de Massas , Modelos Moleculares , Estrutura Terciária de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, we show that one Zn2+ ion in the Zn4 cluster of cyanobacterial metallothionein is inert to exchange with 67Zn2+. We suggest that this is zinc in site A, which together with the surrounding alpha and beta secondary structure forms a zinc-finger fold.
Assuntos
Zinco/química , Ciclotrons , Análise de Fourier , Espectrometria de Massas/métodos , Modelos MolecularesRESUMO
Electron capture dissociation (ECD) of polypeptides has been demonstrated using a commercially available 3 Tesla Fourier transform ion cyclotron resonance (FTICR) instrument. A conventional rhenium filament, designed for high-energy electron impact ionisation, was used to effect ECD of substance P, bee venom melittin and bovine insulin, oxidised B chain. A retarding field analysis of the effective electron kinetic energy distribution entering the ICR cell suggests that one of the most important parameters governing ECD for this particular instrument is the need to employ low trapping plate voltages. This is shown to maximise the abundance of low-energy electrons. The demonstration of ECD at this relatively low magnetic field strength could offer the prospect of more routine ECD analysis for the wider research community, given the reduced cost of such magnets and (at least theoretically) the greater ease of electron/ion cloud overlap at lower field.