The Experts below are selected from a list of 6216 Experts worldwide ranked by ideXlab platform
Natalia Y Tretyakova - One of the best experts on this subject based on the ideXlab platform.
-
Capillary HPLC accurate mass ms ms quantitation of n7 2 3 4 trihydroxybut 1 yl guanine adducts of 1 3 butadiene in human leukocyte dna
Chemical Research in Toxicology, 2013Co-Authors: Dewakar Sangaraju, Colin R Campbell, Melissa Goggin, Peter W Villalta, Maria O Agunsoye, Natalia Y TretyakovaAbstract:1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a Capillary HPLC-high resolution ESI+-MS/MS (HPLC-ESI+-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafilt...
-
quantitative high performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the adenine guanine cross links of 1 2 3 4 diepoxybutane in tissues of butadiene exposed b6c3f1 mice
Chemical Research in Toxicology, 2008Co-Authors: Melissa Goggin, Soobong Park, Christopher P. Anderson, James A Swenberg, Vernon Walker, Natalia Y TretyakovaAbstract:1,3-Butadiene (BD) is an important industrial chemical used in the manufacture of rubber and plastics as well as an environmental pollutant present in automobile exhaust and cigarette smoke. It is classified as a known human carcinogen based on the epidemiological evidence in occupationally exposed workers and its ability to induce tumors in laboratory animals. BD is metabolically activated to several reactive species, including 1,2,3,4-diepoxybutane (DEB), which is hypothesized to be the ultimate carcinogenic species due to its bifunctional electrophilic nature and its ability to form DNA-DNA and DNA-protein cross-links. While 1,4- bis-(guan-7-yl)-2,3,-butanediol ( bis-N7G-BD) is the only type of DEB-specific DNA adduct previously quantified in vivo, four regioisomeric guanine-adenine (G-A) cross-links have been observed in vitro: 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), 1-(guan-7-yl)-4-(aden-3-yl)-2,3-butanediol (N7G-N3A-BD), 1-(guan-7-yl)-4-(aden-7-yl)-2,3-butanediol (N7G-N7A-BD), and 1-(guan-7-yl)-4-(aden-6-yl)-2,3-butanediol (N7G-N (6)A-BD) ( Park ( 2004) Chem. Res. Toxicol. 17, 1638- 1651 ). The goal of the present work was to develop an isotope dilution HPLC-positive mode electrospray ionization-tandem mass spectrometry (HPLC-ESI (+)-MS/MS) method for the quantitative analysis of G-A DEB cross-links in DNA extracted from BD-exposed laboratory animals. In our approach, G-A butanediol conjugates are released from the DNA backbone by thermal or mild acid hydrolysis. Following solid-phase extraction, samples are subjected to Capillary HPLC-ESI (+)-MS/MS analysis with (15)N 3, (13)C 1-labeled internal standards. The detection limit of our current method is 0.6-1.5 adducts per 10 (8) normal nucleotides. The new method was validated by spiking G-A cross-link standards (10 fmol each) into control mouse DNA (0.1 mg), followed by sample processing and HPLC-ESI (+)-MS/MS analysis. The accuracy and precision were calculated as 105 +/- 17% for N7G-N3A-BD, 102 +/- 25% for N7G-N7A-BD, and 79 +/- 11% for N7G-N (6)A-BD. The regioisomeric G-A DEB adducts were formed in a concentration-dependent manner in DEB-treated calf thymus DNA, with N7G-N1A-BD found in the highest amounts. Under physiological conditions, N7G-N1A-BD underwent Dimroth rearrangement to N7G-N (6)A-BD ( t 1/2 = 114 h), while hydrolytic deamination of N7G-N1A-BD to the corresponding hypoxanthine lesion was insignificant. We found that for in vivo samples, a greater sensitivity could be achieved if N7G-N1A-BD adducts were converted to the corresponding N7G-N (6)A-BD lesions by forced Dimroth rearrangement. Liver DNA extracted from female B6C3F1 mice that underwent inhalation exposure to 625 ppm BD for 2 weeks contained 3.1 +/- 0.6 N7G-N1A-BD adducts per 10 (8) nucleotides ( n = 5) (quantified as N7G-N (6)A-BD following base-induced Dimroth rearrangement), while the amounts of N7G-N3A-BD and N7G-N7A-BD were below the detection limit of our method. None of the G-A cross-links was present in control animals. The formation of N7G-N1A-BD cross-links may contribute to the induction of AT base pair mutations following exposure to BD. Quantitative methods presented here may be used not only for studies of biological significance in animal models but potentially to predict risk associated with human exposure to BD.
-
cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor nitrogen mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
-
quantitative analysis of the oxidative dna lesion 2 2 diamino 4 2 deoxy β d erythro pentofuranosyl amino 5 2h oxazolone oxazolone in vitro and in vivo by isotope dilution Capillary HPLC esi ms ms
Nucleic Acids Research, 2006Co-Authors: Brock Matter, Danuta Malejkagiganti, Saari A Csallany, Natalia Y TretyakovaAbstract:A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, Capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding 15N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.
-
cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane
Chemical Research in Toxicology, 2006Co-Authors: Rachel Loeber, Qingming Fang, Mathur Rajesh, And Anthony E Pegg, Natalia Y TretyakovaAbstract:1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-l'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O 6 -alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O 6 -alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32 P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI + -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys 145 , and a neighboring active site residue, Cys 150 . The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys 145 was further confirmed by HPLC-ESI + -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys 145 ). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys 150 was retained. The exact chemical structure of the cross-linked lesion was established as l-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI + -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.
Rachel Loeber - One of the best experts on this subject based on the ideXlab platform.
-
cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor nitrogen mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
-
cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane
Chemical Research in Toxicology, 2006Co-Authors: Rachel Loeber, Qingming Fang, Mathur Rajesh, And Anthony E Pegg, Natalia Y TretyakovaAbstract:1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-l'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O 6 -alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O 6 -alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32 P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI + -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys 145 , and a neighboring active site residue, Cys 150 . The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys 145 was further confirmed by HPLC-ESI + -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys 145 ). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys 150 was retained. The exact chemical structure of the cross-linked lesion was established as l-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI + -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.
-
cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane and antitumor nitrogen mustards
Cancer Research, 2006Co-Authors: Rachel Loeber, Anthony E Pegg, Danae Quirk Dorr, Melissa Goggin, Natalia Y TretyakovaAbstract:5244 O6-alkylguanine DNA alkyltransferase (AGT) repairs O6-alkylguanine lesions in DNA by direct transfer of the O6-alkyl group to an active site cysteine (Cys145), restoring normal guanine and preventing mutagenesis. However, previous studies suggested that AGT expression increased the cytotoxic and mutagenic effects of several bis-electrophiles, including 1,2,3,4-diepoxybutane (DEB) and antitumor nitrogen mustards (NM). Because these bifunctional alkylating agents produce N7-guanine monoadducts that retain one of the electrophilic groups, they are likely to induce DNA-protein cross-links by interacting with nucleophilic residues within AGT. The purpose of this study was to determine the structures of DEB- and NM-induced AGT-DNA conjugates and to identify specific amino acid residues involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant lacking the alkyl acceptor residue. Capillary HPLC-ESI+ mass spectrometric analysis of hAGT that had been incubated with N7-(2’-hydroxy-3’,4’-epoxybut-1’-yl)-deoxyguanosine (dG monoepoxide) or N7-guanine-half mustards of mechlorethamine and chlorambucil revealed the ability of the protein to form either one or two cross-links to dG. The cross-link locations were mapped to Cys145 and Cys150 by HPLC-ESI+-MS/MS analyses of tryptic digests. The same two active site residues participated in cross-linking following the incubation of hAGT with double-stranded DNA in the presence of DEB, mechlorethamine, and chlorambucil. The exact chemical structures of the cross-linked lesions were established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-Gua-BD), N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine (Cys-Gua-EMA), and N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid (Cys-Gua-PBA) by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified proteins analyzed in parallel with synthetic standards. DNA-protein cross-linking is a likely mechanism for cytotoxicity and mutagenicity of DEB and NM in cells expressing AGT protein.
-
interstrand and intrastrand dna dna cross linking by 1 2 3 4 diepoxybutane role of stereochemistry
Journal of the American Chemical Society, 2005Co-Authors: Soobong Park, Rachel Loeber, Christopher P. Anderson, Mahadevan Seetharaman, Roger A JonesAbstract:1,2,3,4-Diepoxybutane (DEB) is a bifunctional electrophile capable of forming DNA-DNA and DNA-protein cross-links. DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then form 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) lesions. All three optical isomers of DEB are produced metabolically from 1,3-butadiene, but S,S-DEB is the most cytotoxic and genotoxic. In the present work, interstrand and intrastrand DNA-DNA cross-linking by individual DEB stereoisomers was investigated by PAGE, mass spectrometry, and stable isotope labeling. S,S-, R,R-, and meso-diepoxides were synthesized from l-dimethyl-2,3-O-isopropylidene-tartrate, d-dimethyl-2,3-O-isopropylidene-tartrate, and meso-erythritol, respectively. Total numbers of bis-N7G-BD lesions (intrastrand and interstrand) in calf thymus DNA treated separately with S,S-, R,R-, or meso-DEB (0.01-0.5 mM) were similar as determined by Capillary HPLC-ESI(+)-MS/MS of DNA hydrolysates. However, denaturing PAGE has revealed that S,S-DEB produced the highest number of interchain cross-links in 5'-GGC-3'/3'-CCG-5' sequences. Intrastrand adduct formation by DEB was investigated by a novel methodology based on stable isotope labeling HPLC-ESI(+)-MS/MS. Meso DEB treatment of DNA duplexes containing 5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5' and 5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5' trinucleotides gave rise to comparable numbers of 1,2-intrastrand and 1,3-interstrand bis-N7G-BD cross-links, while S,S DEB produced few intrastrand lesions. R,R-DEB treated DNA contained mostly 1,3-interstrand bis-N7G-BD, along with smaller amounts of 1,2-interstrand and 1,2-intrastrand adducts. The effects of DEB stereochemistry on its ability to form DNA-DNA cross-links may be rationalized by the spatial relationships between the epoxy alcohol side chains in stereoisomeric N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine adducts and their DNA environment. Different cross-linking specificities of DEB stereoisomers provide a likely structural basis for their distinct biological activities.
Ryszard Lobinski - One of the best experts on this subject based on the ideXlab platform.
-
Capillary HPLC icp ms mapping of selenocompounds in spots obtained from the 2 d gel electrophoresis of the water soluble protein fraction of selenized yeast
Analytical and Bioanalytical Chemistry, 2006Co-Authors: Laure Tastet, Dirk Schaumlöffel, Brice Bouyssiere, Ryszard LobinskiAbstract:A method based on ICP collision-cell MS detection in Capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10-1000 in conditions where the common ES Q-TOF MS detection failed.
-
biosynthesis purification and analysis of selenomethionyl calmodulin by gel electrophoresis laser ablation icp ms and Capillary HPLC icp ms peptide mapping following in gel tryptic digestion
Journal of Analytical Atomic Spectrometry, 2005Co-Authors: Guillaume Ballihaut, Ryszard Lobinski, Laure Tastet, Christophe Pecheyran, Brice Bouyssiere, Olivier F X Donard, Regis GrimaudAbstract:A biosynthesis method was developed to produce a standard of a selenium-containing protein. It consisted of the expression of calmodulin in Escherichia coli culture in the presence of selenomethionine, which allowed the replacement of all methionine residues by selenomethionine. The resulting 17 kDa protein containing 8 selenomethionine residues was purified by two-step hydrophobic interaction chromatography. The selenomethionyl calmodulin was subsequently used to develop a method for the characterization of selenium-containing proteins (detected in the polyacrylamide gel by laser ablation-ICP-MS) by means of peptide mapping using Capillary HPLC-ICP-MS. The monitoring of the 80Se isotope using an ICP mass spectrometer equipped with a collision cell allowed as little as 0.3 pg as Se (1.3 ng ml−1 in the analysed solution) to be detected in the gel. The band containing the protein of interest was excised, the protein was digested with trypsin and the Se-containing peptides were analyzed by Capillary HPLC-ICP-MS. The sensitivity of the method was at least a factor of 5 higher than that of Capillary LC-electrospray MS/MS in similar conditions. Some of the selenopeptides detected by Capillary LC-ICP MS could nevertheless be identified by retention time matching using a set of peptides generated by trypsin digestion from the concentrated selenomethionyl calmodulin standard.
-
determination of selenomethionine and selenocysteine in human serum using speciated isotope dilution Capillary HPLC inductively coupled plasma collision cell mass spectrometry
Analytical Chemistry, 2004Co-Authors: Jorge Ruiz Encinar, Yasumitsu Ogra, Dirk Schaumlöffel, Ryszard LobinskiAbstract:A method for the accurate determination of selenoamino acids in human serum by HPLC−ICPMS was developed using the species-specific isotope dilution analysis principle. A serum sample was enzymatically digested with a mixture of lipase and protease after derivatization of the selenocysteine residues with iodoacetamide. The selenoamino acid fraction was isolated by size exclusion LC followed by the separation of selenomethionine and the carboxymethylated selenocysteine by Capillary HPLC. The isotope-specific determination of 77Se and 80Se was achieved on-line by ICP collision cell MS allowing the removal of polyatomic interferences. Quantification was carried out by isotope dilution using a 77Se-labeled selenomethionine spike and the determination of the 77Se/80Se ratio in the cHPLC selenomethionine peak. The accurately determined selenomethionine was used as an internal standard for the selenocysteine determination from the same chromatogram. The modification of the previously developed cHPLC−ICPMS interfa...
Joanna Szpunar - One of the best experts on this subject based on the ideXlab platform.
-
characterization of selenium incorporation into wheat proteins by two dimensional gel electrophoresis laser ablation icp ms followed by Capillary HPLC icp ms and electrospray linear trap quadrupole orbitrap ms
Analytical Chemistry, 2013Co-Authors: Juliusz Bianga, Espen Govasmark, Joanna SzpunarAbstract:A method has been developed for a rapid and precise location of selenium-containing proteins in large two-dimensional (2D) electrophoresis gels. A sample was divided into four aliquots which were analyzed in parallel by 1D isoelectric focusing electrophoresis (IEF)–laser ablation (LA) inductively coupled plasma mass spectrometry (ICP MS), 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)–LA ICP MS, and, in duplicate, by 2D IEF-PAGE. On the basis of the 1 D electropherograms obtained, areas supposed to contain the largest concentrations of Se were subjected to LA ICP MS imaging to locate precisely the position of Se-containing proteins which were then identified in the parallel 2D gel by electrospray Orbitrap MS/MS. The method was applied to the identification and semiquantitative determination of selenium storage proteins in wheat. MS evidence is presented for the Se–S substitution in plants not only in methionine but also in cysteine.
-
icp ms assisted identification of selenium containing proteins in 2d gels using a new Capillary HPLC icp ms interface and orbitrap tandem mass spectrometry
Journal of Analytical Atomic Spectrometry, 2013Co-Authors: Juliusz Bianga, Joanna SzpunarAbstract:An improved instrumental approach is discussed for the identification of Se-containing proteins separated by 2D polyacrylamide gel electrophoresis. A protein, detected in a gel by laser ablation ICP-MS 2D 78Se imaging, was excised from another gel obtained in parallel under identical conditions, and digested with trypsin. Selenium-peptides were detected by Capillary HPLC–ICP MS using a new robust commercial interface and an ICP mass spectrometer fitted with a frequency-matching RF generator facilitating work at high concentrations of acetonitrile in the mobile phase. The identification of selenopeptides was carried out by means of hybrid linear quadrupole trap-Orbital trap MS offering a higher intrascan dynamic range and better mass accuracy than the hitherto used Q-TOF (time-of-flight) mass spectrometers. An example of the identification of Se-containing glyceraldehyde-3-phosphate dehydrogenase-3 in yeast is discussed.
-
quantification of a selenium containing protein in yeast extract via an accurate determination of a tryptic peptide by species specific isotope dilution Capillary HPLC icp ms
Chemia Analityczna, 2005Co-Authors: Aleksandra Polatajko, Dirk Schaumlöffel, Jorge Ruiz Encinar, Joanna SzpunarAbstract:A method based on the species-specific isotope dilution analysis was developed for the accurate determination ofanAsp-Tyr-SeMet-Gly-Ala-Ala-Lys peptide directly in a tryptic digest of an aqueous extract ofselenized yeast.For this purpose a 7 7 Se labeled peptide to be used as a standard had been purified by 2D liquid chromatography from yeast grown on 7 7 SeO 3 -rich culture and quantified by reversed-phase isotope dilution analysis. The sample mixed with the 7 7 Se-labeled peptide spike was analyzed by Capillary HPLC-ICP collision cell MS. The isotopic labeling allowed the unequivocal identification of the peptide among all the Se species detected. It compensates for retention time shift and possible peak distortion due to the injection of a complex salt-rich matrix onto a Capillary column. The isotope ratio of selenium ( 7 7 Se/ 8 0 Se) was measured in the peak corresponding to the peptide of interest enabling its accurate quantification by isotope dilution. The determined concentration of the peptide, which was quantitatively ferried from a selenized 12 kDa heat-shock protein, made the quantification ofthe latter possible by Capillary HPLC-ICP MS directly in a yeast extract, without any additional purification.
Richard Wilson - One of the best experts on this subject based on the ideXlab platform.
-
comparative proteomic analysis of normal and collagen ix null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen ix interactome
Journal of Biological Chemistry, 2013Co-Authors: Bent Brachvogel, Frank Zaucke, Manuel Koch, Jeffrey J. Gorman, Münire Dayakli, Emma L. Norris, Keyur A. Dave, Jacek Stermann, John F Bateman, Richard WilsonAbstract:The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by Capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.
-
Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome
The Journal of biological chemistry, 2013Co-Authors: Bent Brachvogel, Frank Zaucke, Manuel Koch, Jeffrey J. Gorman, Münire Dayakli, Emma L. Norris, Keyur A. Dave, Jacek Stermann, Richard WilsonAbstract:BACKGROUND : Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS : Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION : Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE : We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by Capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.
