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Selected AbstractsBJ46a, a snake venom metalloproteinase inhibitorFEBS JOURNAL, Issue 10 2001Isolation, characterization, cloning, insights into its mechanism of action Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4 -reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition. [source] Differentiation of sulfate and phosphate by H/D exchange mass spectrometry: application to isoflavoneJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2004Akira Kanakubo Abstract Often phosphorylation or sulfation is an important step which occurs in the signal transduction and cascade of metabolic pathways. Some natural products and metabolites contain one or more sulfate or phosphate groups. Isoflavone sulfate has been identified from high-resolution mass spectrometry (HRMS) and enzymatic digestion by sulfatase. We previously reported the new water-soluble isoflavone analogs, daidzein 7- O -phosphate and genistein 7- O -phosphate, which were surprisingly hydrolyzed by sulfatase. In this previous study, we could not determine the phosphate from the results of HRMS and enzymatic digestion, that is, HRMS and enzymatic digestion did not provide clear evidence. In this case, we drew conclusions from NMR analysis. HRMS has been ineffective with a regular fast atom bombardment (FAB) mass spectrometer to distinguish between phosphate and sulfate since the mass difference is only 0.009 mass units. There was, however, no conventional method of microanalysis to distinguish phosphate from sulfate owing to the same nominal mass. It is still very difficult to determine by negative FABMS [OP(O)(OH)2] = 80 and [OS(O)2OH] = 80. In this paper, we report a method to distinguish between these groups by using a popular low-resolution mass instrument; thus, phosphate and sulfate were measured by H/D exchange mass spectrometry at the picomole level to differentiate [OP(O)(OD)2] = 82 and [OS(O)2OD] = 81, respectively. This method is applicable not only to the isoflavone, but also to other phospho and sulfo compounds. Copyright © 2004 John Wiley & Sons, Ltd. [source] EU Forum: The CREATE Project: development of certified reference materials for allergenic products and validation of methods for their quantificationALLERGY, Issue 3 2008R. Van Ree Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project. [source] In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006Brigitte L. Simons We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100,mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the , -amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b -ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9 - to D9 -trimethyl-labeled peptides of , -casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd. [source] Quantification of group 5 grass pollen allergens in house dustCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000B. Fahlbusch Background It is widely known and accepted that grass pollen is a major outdoor cause of hay fever. However, it is of virtual importance for grass pollen allergic patients with symptoms all the year round to know the concentration of grass pollen allergens in their homes. Objective The main objective of this study was to quantify the amount of grass pollen allergen in mass units (,g Phl p 5) in dust settled indoors and to detect the distribution of allergenic activity in different sampling locations of homes. Furthermore, we studied the seasonal fluctuation of allergen content in dust samples. Methods We adapted the two site binding assay for detection of group 5 grass pollen allergens in samples from randomly selected homes in Hamburg (n = 371), Erfurt (n = 396), Hettstedt (n = 353), Zerbst (n = 289) and Bitterfeld (n = 226), Germany. Dust samples were collected from floor of living room (LR), bedroom (BR) or children's room (CR) and mattress (MA) during period of June 1995 to August 1998. The amount of the major grass group 5 allergens was detected in ,g/g dust. Results Phl p 5 was detected in 67% of the samples analysed (n = 4760). The range was between undetectable (< 0.03 ,g/g dust) and 81 ,g/g dust. Phl p 5 levels were significantly higher in the dust from LR (geometric mean 0.117 ,g/g dust) or BR/CR floors (geometric mean 0.098 ,g/g dust) than in mattresses (geometric mean 0.043 ,g/g dust). We observed seasonal fluctuation of indoor Phl p 5 levels with peak in June but also annual differences. Thus Phl p 5 content indoors reflects also the different quantities of pollen counts of annual courses. During pollination period we found two times higher Phl p 5 levels (0.172 ,g/g dust, P < 0.001) than outside of grass pollination season (0.095 ,g/g dust). The indoor Phl p 5 levels outside of season seem to be independent of pollination before. We suppose that settled pollen grains or allergenic material from outdoor particles carried indoors via footwear and clothes accumulates in house dust. Conclusion Although we not known how the allergens in settled dust are equilibrated with those in the air, the considerable high level of Phl p 5 in indoor dust even during periods when no grass pollen is present in the atmosphere may be an important cause of pollen-allergy symptoms outside of season. [source] | |||||||||||||