- United Kingdom
Research Interests:
Research Interests: Chemistry, Proteomics, Immunohistochemistry, Medicine, Biological Sciences, and 15 moreSoftware, Humans, Smooth muscle, Proteins, Protein Expression, Gel electrophoresis, Cell Proliferation, SAPHENOUS VEIN, Amino Acid Sequence, Sodium Dodecyl Sulphate, Polyacrylamide Gel Electrophoresis, QD, Isoelectric point, Differential expression, and Medical and Health Sciences
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli... more
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.
Research Interests: Biochemistry, Molecular Biology, Biology, Medicine, Humans, and 15 moreEscherichia coli, Biochemical, Affinity chromatography, Enzyme, Isoforms, Recombinant DNA, Isoenzymes, Amino Acid Profile, Molecular Mass, Metabolic pathway, Amino Acid Sequence, Fusion Protein, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
Research Interests: Dentistry, Immunology, Medical Microbiology, Biology, Autoimmunity, and 15 moreGene Silencing, Biological Sciences, Fibrinogen, Humans, Genetically modified organisms, Arthritis, Clinical Sciences, High Pressure Liquid Chromatography, CHEMICAL SCIENCES, Amino Acid Sequence, Citrulline, DNA binding proteins, Glycosyl Hydrolases, Medical and Health Sciences, and Citrullination
Research Interests:
Research Interests: Chemistry and Riboflavin
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. If the... more
Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. If the ionization regime deposits sufficient excess energy, a proportion of the sample molecules will dissociate, the pattern of product ions formed being dependent on the structure of the intact compound (Fig. 1). A mass spectrum thus consists of the masses (strictly mass to charge ratios, m/z) of these ions plotted against abundance. Interpretation of the spectrum thus affords information about both the mol wt and the structure of the sample. By the standards of most other physical methods, mass spectrometry is fairly sensitive, requiring somewhere between low picomoles and nanomoles of material, depending on the ionization method employed, but against this must be set its destructive nature. The present introduction aims to provide a brief overview of the technique, to define some of the key terms, and to offer a short tour of some of the different instruments that are more or less legitimately called mass spectrometers. Readers wishing a more detailed account should consult refs. 1-9. Arecent volume of Methods in Enzymology (5) devoted entirely to mass spectrometry is particularly recommended, since both instrumentation and applications are comprehensively covered.
Research Interests:
Research Interests: Chemistry, Computational Biology, Biotechnology, Polysaccharides, Medicine, and 15 moreErythropoietin, Humans, Mice, Animals, Recombinant DNA, Protein Conformation, Protein isoforms, Biological activity, Recombinant Proteins, Biological Assay, Carbohydrate Sequence, Molecular Sequence Data, Medical and Health Sciences, Cardiovascular medicine and haematology, and Cell culture techniques
Research Interests: Molecular Biology, Mass Spectrometry, Biology, Proteomics, Leishmania, and 13 moreMedicine, Biological Sciences, Molecular, Animals, Host Parasite Interaction, Proteome analysis, Protozoan Proteins, Two-Dimensional Gel Electrophoresis, Two dimensional Gel Electrophoresis, Proteome, Gene Expression Regulation, Infectivity, and Medical and Health Sciences
Research Interests: Chemistry, Biology, Glycobiology, Leishmania, Magnetic Resonance Spectroscopy, and 13 moreMedicine, Mammals, Biological Sciences, Phylogeny, Methylation, Lizards, Animals, Stereochemistry, Glucans, Structural Similarity Index, Nuclear Magnetic Resonance Spectroscopy, Structural similarity, and Medical and Health Sciences
Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been... more
Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.
Research Interests:
ABSTRACT
Research Interests: Biochemistry, Chemistry, Protein Folding, Kinetics, Medicine, and 10 moreHumans, Escherichia coli, Animals, Amino Acid Sequence, Recombinant Proteins, Hydrogen-Ion Concentration, Dissociation Constant, Biochemistry and cell biology, Matrix Metalloproteinase, and Medical biochemistry and metabolomics
Research Interests:
Research Interests:
Research Interests:
Research Interests:
... The phylum “Deinococcus/Thermus” represents an ancient line of descent within the domain Bacteria that includes the extremely radiation-resistant bacteria of the genus Deinococcus (Battista and Rainey, 2001; Rainey et al., 2005), and... more
... The phylum “Deinococcus/Thermus” represents an ancient line of descent within the domain Bacteria that includes the extremely radiation-resistant bacteria of the genus Deinococcus (Battista and Rainey, 2001; Rainey et al., 2005), and the slightly thermophilic or thermophilic ...
Research Interests:
Picolinyl esters have been recently introduced for the mass spectrometric structure determination of unsaturated and cyclopropane fatty acids. We have used these derivatives to characterize the unsaturated and cyclopropane acids of... more
Picolinyl esters have been recently introduced for the mass spectrometric structure determination of unsaturated and cyclopropane fatty acids. We have used these derivatives to characterize the unsaturated and cyclopropane acids of Campylobacter species. The principle unsaturated acids were shown to be 11‐octadecenoic acid, with smaller amounts of 9‐hexadecenoic acid, 9‐octadecenoic acid and 9, 11‐octadecadienoic acid. The only cyclopropane acid present was cis 11, 12‐methylene octadecanoic acid. There was no interspecific variation in the position of unsatu‐ration or methylene bridges, even in the recently discovered gastric Campylobacter like organisms (GCLO) or the urease positive thermophilic Campylobacters (UPTC), the fatty acid profiles of which were consistent with their inclusion in the genus Campylobacter.
Research Interests:
Research Interests:
... Jun Yan, Robin Wait*, Tom Berkelman#, Rachel Harry, Jules Westbrook, Colin Wheeler and Mike Dunn. Heart Science Center, Harefield Hospital, Imperial College School of Medicine, Hill End Road, Harefield, UB9 6JH, *Kenndy Institute of... more
... Jun Yan, Robin Wait*, Tom Berkelman#, Rachel Harry, Jules Westbrook, Colin Wheeler and Mike Dunn. Heart Science Center, Harefield Hospital, Imperial College School of Medicine, Hill End Road, Harefield, UB9 6JH, *Kenndy Institute of Rheumatology, Hammersmith ...
Research Interests:
Research Interests:
Research Interests:
The pathological importance of tumor necrosis factor (TNF)-alpha in rheumatoid arthritis (RA) is now widely accepted. Ex vivo data from synovial cell cultures suggest that direct cell contact between activated T-cells and macrophages may... more
The pathological importance of tumor necrosis factor (TNF)-alpha in rheumatoid arthritis (RA) is now widely accepted. Ex vivo data from synovial cell cultures suggest that direct cell contact between activated T-cells and macrophages may be an important driver of macrophage TNF-alpha production in the RA joint. However, the ligand/receptor pairs driving this cell contact signal remain obscure. One reason for this is that plasma membrane (PM) proteins are resistant to systematic analysis using traditional proteomic approaches. In this chapter we present a method for the enrichment and resolution of PM proteins from murine T-cell hybridomas as a prelude to identification by tandem mass spectrometry. We used cell surface biotinylation, differential centrifugation and subsequent streptavidin affinity capture, followed by solution phase iso-electric focussing and tandem mass spectrometry to identify 75 PM proteins and make semiquantitative comparisons of resting and activated cells. The method is applicable to a wide variety of cell types.
Research Interests:
Purpose: Fibronectin is an extracellular matrix (ECM) molecule implicated in a number of processes during physiology and inflammation. Expression of full-length FN is upregulated in chronic conditions such as rheumatoid arthritis and... more
Purpose: Fibronectin is an extracellular matrix (ECM) molecule implicated in a number of processes during physiology and inflammation. Expression of full-length FN is upregulated in chronic conditions such as rheumatoid arthritis and osteoarthritis and fibronectin fragments induce specific biological effects. The aggrecanases (ADAMTSs) and MMPs are believed to be the key enzymes mediating joint damage in arthritis. The C-terminal heparin-binding region of fibronectin has been implicated in regulating aggrecanase activity in vitro, but the activation of aggrecanase activity by this region has not been investigated in experimental models of arthritis. Methods: The expression of fibronectin and its fragments was investigated in normal and arthritic tissue by Western blotting. Normal porcine articular cartilage was then used to test recombinant fragments of fibronectin, either alone, or in combination with the cytokines IL-1 and TNF, for their ability to induce aggrecanase and MMP activ...
Research Interests:
Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1)more
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2... more
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant ...
Research Interests: Biology, Membrane Proteins, Medicine, Biological Sciences, Humans, and 13 moreBiochemical, CHEMICAL SCIENCES, RNA-binding proteins, HeLa cells, Isoenzymes, Tristetraprolin, Base Sequence, Structure activity Relationship, DNA binding proteins, Molecular Sequence Data, DNA mutational analysis, conserved sequence , and Medical and Health Sciences
Text Character count for abstract text: 2225 (525 Characters Remaining) Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disease characterised by inflammation followed by tissue rebuilding or fibrosis. Failure by the body to... more
Text Character count for abstract text: 2225 (525 Characters Remaining) Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disease characterised by inflammation followed by tissue rebuilding or fibrosis. Failure by the body to effectively regulate inflammation is a hallmark of RA. It has been suggested that periodontal disease is one mechanism whereby tissue inflammation is triggered in RA. One of the organisms implicated in periodontal disease: Porphorymonas gingivalis, is an anaerobic pathogen that is known to produce peptidyl arginine demiminase (PAD), the only known bacterial PAD which causes citrullination. Cleavage of extracellular matrix (ECM) substrates in RA is known to lead to the production of ECM damage-associated molecular patterns, or DAMPs that can then be available for citrullination, thereby mediating chronic inflammation in RA.
Research Interests:
ABSTRACT Prokaryotes possess an enormous variety of polar lipids, primarily phospholipids and glycolipids, that cannot be easily identified at the chemical level. However, by using thin-layer chromatography, these polar lipids have been... more
ABSTRACT Prokaryotes possess an enormous variety of polar lipids, primarily phospholipids and glycolipids, that cannot be easily identified at the chemical level. However, by using thin-layer chromatography, these polar lipids have been shown to constitute important chemotaxonomic parameters that can aid in the characterization of new taxa. The analysis of polar lipids is recommended for the description of new taxa because these lipids can be used to confirm the taxonomic affiliation of many taxa. This chapter is devoted to the methods used in polar lipid analysis, including the growth of the organisms under standardized conditions, and the methods for extraction and visualization of the lipids by thin-layer chromatography and for the identification of some of the properties of the lipids by using colourimetric spray reagents. Examples of the value of mass spectrometry for the identification of the lipids are also presented. © 2011 Elsevier Ltd.
Research Interests:
ABSTRACT The fatty acid composition is probably the most important chemotaxonomic parameter used for the differentiation of prokaryote taxa at the species level because it correlates very well with the accepted DNA:DNA hybridization-based... more
ABSTRACT The fatty acid composition is probably the most important chemotaxonomic parameter used for the differentiation of prokaryote taxa at the species level because it correlates very well with the accepted DNA:DNA hybridization-based species concept of many groups of bacteria. However, care must be taken to standardize the conditions of growth as well as those of the gas chromatography. If these standardized conditions are followed, valuable information may be acquired from the cellular fatty acids composition. This chapter describes methods that should be applied and the care that should be taken to obtain uniform values from fatty acid determination. © 2011 Elsevier Ltd.
Research Interests:
Isolated rat hearts or isolated ventricular myocytes were treated with H 2 O 2 (1μM-10mM). This caused a dose-dependent increase in interprotein disulfide bond formation between PKAs two regulatory RI subunits observed on non-reducing... more
Isolated rat hearts or isolated ventricular myocytes were treated with H 2 O 2 (1μM-10mM). This caused a dose-dependent increase in interprotein disulfide bond formation between PKAs two regulatory RI subunits observed on non-reducing immunoblots. Control myocytes contain 14% disulfide PKA dimer, increasing to 68% (p 2 O 2 (100μM, 5min). We hypothesised this may have a functional correlate. Fractionation showed the RI disulfide translocated from cytosol to membrane and myofilament/nuclear enriched fractions. This was confirmed by confocal immunofluorescent imaging of RI in myocytes, with enhanced filament and nuclear staining only present in H 2 O 2 cells. H 2 O 2 also increased phosphorylation of multiple proteins, including a 25kDa (4.5 fold, p 2 O 2 (100μM) treatment in cardiac myocytes was increased amplitude of contraction to 0.204μm, compared to 0.111μm in controls (p 2 O 2 did not elevate cAMP at any dose (1μM-1mM). H 2 O 2 -induced substrate phosphorylation and increased contraction was blocked by the PKA inhibitor H89 (10μM). H 2 O 2 also caused a dose dependent decrease in Type I PKA holoenzyme complex size, as evidenced by immunoblot analysis of the catalytic subunit in fractions eluting from a Superose 12 gel filtration column, further supporting activation without cAMP. PKA R subunits (which have AKAP binding sites) were affinity purified from hearts with cAMP-agarose and analysed by Coomassie stained SDS-PAGE. One protein was present only in H 2 O 2 samples and identified by LC-MS/MS as α myosin heavy chain (αMHC). This interaction was confirmed by immunoprecipitating αMHC, which co-purified with RI PKA in a H 2 O 2 dose dependent manner. In vitro binding assays demonstrated the interaction only occurred when RI was in the disulfide oxidised state. We conclude that Type I PKA is redox sensitive and is activated directly by H 2 O 2 by a mechanism involving interprotein disulfide formation, with αMHC acting as an AKAP to target it to myofilament substrates.
Research Interests:
<b>Copyright information:</b>Taken from "Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis"Arthritis Research &amp; Therapy... more
<b>Copyright information:</b>Taken from "Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis"Arthritis Research &amp; Therapy 2006;8(1):R23-R23.Published online 3 Jan 2006PMCID:PMC1526559. Membrane extracts isolated from IL-1 plus oncostatin M stimulated chondrocytes were treated with or without FP-biotin. Labelled proteinases were isolated using streptavidin-agarose beads and eluted with reducing SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE and stained with colloidal Coomassie. The 97 kDa protein was identified by mass spectrometry to be FAPα.
Research Interests:
The profiling of cellular lipids by GC/MS is a valuable technique for the identification and classification of micro-organisms. Most procedures however entail the hydrolysis and derivatization of complex lipids prior to analysis. Fast... more
The profiling of cellular lipids by GC/MS is a valuable technique for the identification and classification of micro-organisms. Most procedures however entail the hydrolysis and derivatization of complex lipids prior to analysis. Fast atom bombardment mass spectrometry avoids these stages and enables the direct analysis of unmodified polar lipids. By the use of collisional activation and linked scanning, complex mixtures of phospholipids can be characterized without the necessity of a chromatographic separation step, and the disposition of substituents can be established by massmeasurement of the products of phospholipase digestion. This chapter describes the application of these techniques to problems of microbiological identification, classification and physiology.
Research Interests:
Research Interests:
Research Interests:
Plasma membrane (PM) proteins are of particular interest to cell biologists because of their role in transducing information from the external environment to the cell interior, and because of their potential as therapeutic targets. The... more
Plasma membrane (PM) proteins are of particular interest to cell biologists because of their role in transducing information from the external environment to the cell interior, and because of their potential as therapeutic targets. The hydrophobicity and large size of these proteins renders their analysis by conventional proteomic approaches using 2D-electrophoresis problematic, limiting our ability to evaluate alterations of cell surface architecture as a function of varying physiological, pathological, or developmental state.In this chapter, we describe a simple method for enrichment and separation of plasma membrane proteins, prior to their identification by tandem mass spectrometry. Cell surface proteins are labeled with biotin using a reagent which does not enter the cell, purified by differential centrifugation and then affinity captured with streptavidin-agarose beads, before separation by a combination of solution-phase isoelectric focusing, and gradient gel electrophoresis,...
Research Interests:
Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that... more
Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage...
Research Interests: Chemistry, Immunology, Cytokines, Immunohistochemistry, Gene expression, and 15 moreExtracellular Matrix, Humans, Experimental Pathology, Cartilage, Animals, Arthritis, Clinical Sciences, Cattle, Active site, Articular Cartilage Wear, Chondrocytes, Cell Surface Markers, Articular Cartilage, Gene Expression Regulation, and Cell Membrane
Nonreducing two-dimensional gel electrophoresis (2-DE) is described for the study of immunoglobulin disorders with asynchronous production of single chains. Unlike classical reducing 2-DE, this method can distinguish between complex... more
Nonreducing two-dimensional gel electrophoresis (2-DE) is described for the study of immunoglobulin disorders with asynchronous production of single chains. Unlike classical reducing 2-DE, this method can distinguish between complex intact molecules and their free single chains (with different degrees of polymerization) and will thus be helpful for diagnosis of this type of disease. Examples are taken from canine patients, but the method may also be applied to both urine and serum specimens from other species. Nonreducing 2-DE thus represents a useful tool complementary to classical 2-DE, when further information about the appearance of free subunits or modifications of proteins are required, even in the presence of intact molecules.
Research Interests:
Research Interests: Chemistry, Chromatography, Proteomics, Medicine, Biological Sciences, and 11 moreCerebrospinal Fluid, Transthyretin, Sheep, Animals, Precipitation, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Electrophoresis, Rats, Two-Dimensional Gel Electrophoresis, Two dimensional Gel Electrophoresis, and Medical and Health Sciences
Research Interests: Bipolar Disorder, Mass Spectrometry, Proteomics, Medicine, Prefrontal Cortex, and 15 moreMolecular Psychiatry, Biological Sciences, Humans, Female, Male, Middle Aged, Molecular Characterization, Adult, Analysis of Variance, Functional Group, Proteome analysis, Case Control Studies, Dorsolateral Prefrontal Cortex, Differential expression, and Medical and Health Sciences
Research Interests:
Research Interests:
Research Interests: Mass Spectrometry, Biology, Oxidative Stress, Medicine, Molecular, and 15 moreAnimals, Polymerase Chain Reaction, Glutathione, Phosphorylation, Active site, Peroxidases, Methionine, Heat Shock, Molecular Mass, Heat Shock Protein, Amino Acid Sequence, Gene Expression Regulation, Peroxiredoxins, Molecular Sequence Data, and Cardiovascular medicine and haematology
Research Interests: Chemistry, Proteomics, Oxidative Stress, Biological Chemistry, Medicine, and 13 moreBiological Sciences, Animals, Male, Biological, Proteins, CHEMICAL SCIENCES, Rats, Wistar Rats, Two-Dimensional Gel Electrophoresis, Heart Ventricles, Oxidation-Reduction, Immunoblotting, and Medical and Health Sciences
Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Animals, and 14 moreMale, Biological, Heart, Phosphorylation, CHEMICAL SCIENCES, Hydrogen Peroxide, Rats, Wistar Rats, Oxidation-Reduction, Oxidants, Protein Transport, Disulfide bond, Medical and Health Sciences, and In Vitro Techniques
Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates... more
Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as alpha-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated alpha-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%)...