Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry

A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.     

Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli

DNA substrates containing O⁶-n-butylguanine, O⁶-iso-butylguanine,O⁶-n-propylguanine and O⁶-iso-propylguanine were prepared byreaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea.These substrates were used to test the ability of O⁶-alkylguanine-DNAalkyltransferases from Escherichia coli and rat liver to removesuch alkyl groups from the O⁶-position of guanine. It was foundthat all of these adducts were removed by the alkyltransferases,but the branched alkyl chain iso-butyl- and iso-propyl adductswere removed very slowly. Also, when tested with a DNA substratecontaining both O⁶-n-propylguanine and O⁶-iso-propylguanine,the alkyltransferases removed almost all of the n-propyl-adductbefore the iso-propyl-adduct was attacked. Both alkyltransferasesshowed a decreasing rate of reaction as the size of the alkylgroup increased, but there was a significant difference betweenthe rat liver and E. coli alkyltransferase in the relative rates.The rat liver alkyltransferase repaired O⁶-methylguanine moreslowly than the E. coli protein, but was considerably more rapidthan the bacterial equivalent when acting on n-propyl- and n-butyl-adducts.The relative rates of repair were methyl > ethyl > n-propyl> n-butyl >iso-propyl, iso-butyl for the E. coli alkyltransferaseand methyl > ethyl, n-propyl > n-butyl > iso-propyl,iso-butyl > 2-hydroxyethyl for the rat liver protein. Theseresults indicate that differential rates of repair may contributeto the relative risks of carcinogenesis and mutagenesis by exposureto alkylating agents of different size and that rates of repairmay be species specific and must be determined from specificmeasurements rather than extrapolated from data on other organisms.     

亲和纯化技术联合质谱分析筛选乳腺癌中p90核糖体S6蛋白激酶4相互作用蛋白

背景与目的：p90核糖体S6蛋白激酶4基因(ribosomal S6 kinase 4 gene,RSK4)作为抑癌基因,能够抑制细胞增殖、迁移并诱导细胞的凋亡,但与其配合发挥相应功能的相互作用蛋白尚不明确。该研究利用Flag标签纯化技术和质谱分析筛选鉴定乳腺癌细胞株MDA-MB-231中的RSK4相互作用蛋白。方法：构建含有RSK4全长和Flag亲和标签的真核表达载体pcDNA3.1/EGFP-RSK4-Flag,将其转染至MDA-MB-231乳腺癌细胞株,72 h后收集细胞蛋白。采用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain re-action,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测RSK4表达情况。运用标签分离纯化RSK4蛋白,液相质谱/质谱分析技术(liquid chromatography mass spectrometry/mass spectrometry,LC-MS/MS)鉴定RSK4相互作用蛋白,通过生物信息学的方法(GO分析和IPA分析)归纳分析互作蛋白以及与RSK4相互作用机制。结果：通过标签纯化联合质谱分析成功鉴定出丝氨酸/苏氨酸蛋白激酶38(serine/threonine-protein kinase 38,STK38)/丝氨酸/苏氨酸蛋白激酶38类(serine/threonine-protein kinase 38-like,STK38L)、MOB激酶致活因子2(MOB kinase activator 2,MOB2)、蛋白精氨酸N甲基转移酶5(protein arginineN-methyltransferase 5,PRMT5)等24个RSK4相互作用蛋白。对所鉴定出的互作蛋白进行生物信息学分析,提示RSK4互作蛋白组参与包含细胞凋亡和细胞迁移运动等在内的多种生物信号通路。结论：利用亲和纯化技术联合质谱鉴定成功筛选出RSK4相互作用蛋白并通过生物信息学分析获得了RSK4参与乳腺癌细胞的凋亡、迁移相关生物调控网络。     

Identification of signature volatiles to discriminate Candida albicans,glabrata, krusei and tropicalis using gas chromatography and mass spectrometry

Oral candidiasis is the most frequent fungal infection of the oral cavity. Clinical diagnoses require mycological confirmation, which is time‐consuming in case of culture testing. The aim of the study was to identify signature volatiles to develop a chairside breath test to diagnose oral candidiasis. Headspaces above Candida albicans, glabrata, tropicalis, krusei cultures, and growth media as control were analysed after eight and 24 h using offline gas chromatography and mass spectrometry. The identification of signature volatiles was assisted using various microbial databases. Retrieved volatile patterns enabled Candida species discrimination in vitro. For C. albicans 3‐methyl‐2‐butanone and styrene and for C. krusei a combination of p‐xylene, 2‐octanone, 2‐heptanone and n‐butyl acetate were found to be specific. 1‐hexanol was found in C. tropicalis, but is emitted by a variety of other microorganisms. C. glabrata was characterised through the absence of these volatiles. The development of a breath test is a promising approach in confirming suspicions of oral candidiasis. To confirm the retrieved results in vivo, breath tests in affected and healthy subjects have to be performed.     

Characterization of an N6-oxopropenyl-'-deoxyadenosine adduct in malondialdehyde-modified DNA using liquid chromatography/electrospry ionization tandem mass spectrometry

Malondialdehyde (MDA), a product of lipid peroxidation, causesmutations in bacterial and mammalian cells and cancer in rats.MDA reacts with deoxynucleosides in vitro and the monomericadduct of MDA with deoxyguanosine (M₁G-dR) is the major adductM₁G-dR has been detected in rat and human liver. Random mutagenesisstudies with MDA-modified DNA and recent ³²P-postlabeling studiesindicate that in addition to M₁G-dR, adducts to deoxyadenosinemay also be formed. We have utilized liquid chromatography coupledwith electrospray ionization tandem mass spectrometry to characterizean N⁶-oxopropenyl-2'-deoxyadenosine adduct (M₁A-dR) in calfthymusDNA modified with MDA.     

Measurement of MeIQx and DiMeIQx in fried beef by capillary column gas chromatography electron capture negative ion chemical ionisation mass spectrometry

A gas chromatographic—mass spectrometric assay has beendeveloped for the simultaneous measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) and 2-amino-3,4,8-dimethylimidazo[4,5-f]quinoxaline(DiMeIQx) in fried beef. The method employs capillary columngas chromatography, electron capture negative ion chemical ionisationmass spectrometry and a stable isotope labelled analogue ofMeIQx (the synthesis of which is described) as common internalstandard. Two patties of lean minced beef which had been cookedseparately were analysed and found to contain both compounds(patty 1–2.4 ng MeIQx/g meat, 1.2 ng DiMeIQx/g meat; patty2–1.3 ng MeIQx/g meat, 0.5 ng DiMeIQx/g meat). Neithercompound was present in the meat prior to cooking.     

Determination of vinca alkaloids in human plasma by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

 A sensitive assay was developed for the quantitation of vinblastine, desacetylvinblastine and vincristine using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Analyses were performed on an Ultrasphere C₁₈ microbore column using ammonium acetate as mobile phase. The calibration curves were linear across the range of 0.51–4.00 ng/ml (0.63–4.93 nM) for vinblastine, 0.74–3.93 ng/ml (0.96–5.11 nM) for desacetylvinblastine and 0.30–3.95 ng/ml (0.36–4.79 nM) for vincristine. Vinca alkaloid concentrations were measured with an accuracy and precision within 11%. This assay could be implemented to determine the plasma concentrations for pharmacokinetic studies of vinblastine, desacetylvinblastine and vincristine in conjunction with clinical trials. Received: 15 December 1995 / Accepted: 16 June 1996     

Repair of synthetic oligonucleotides containing O6-methylguanine, O6-ethylguanine and O4-methylthymine, by O6-alkylguanine-DNA alkyltransferase

32P-Labelled self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, and O4-methylthymine have been synthesized and used as substrates for the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AAT). The reaction was second-order with rate constants of 2.6 X 10(7) M-1 sec-1 for O6-methylguanine, 2.6 X 10(4) M-1 sec-1 for O6-ethylguanine and 2.5 X 10(3) M-1 sec-1 for O4-methylthymine. These oligomers should allow sensitive and specific assay of the mammalian enzyme.     

Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine, and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase

Self-complementary oligodeoxynucleotides have been synthesizedcontaining O⁶-methylguanine (O⁶meG), O⁶-ethylguanine (O⁶etG),O⁶-isopropylguanine (O⁶iprG) and O⁴-methylthymine (O⁴meT). Theyanneal in solution to give double-stranded DNA. These doublehelices have been used as substrates for the DNA repair proteinO⁶-alkyl-guanine-DNA-alkyltransferase coded for by the ada geneof Escherichia coli. The repair followed second-order chemicalkinetics. O⁶meG was repaired by the 19-kd transferase at a rateof 2.54x10⁷ M^–1 s^–1 which is close to the theoreticallimit for a diffusion-controlled reaction; O⁶etG and O⁴meT arerepaired 1000 and 10000 times more slowly. The 39-kd alkyltransferase(which is precursor to the 19-kd form) and the 19-kd transferaserepaired O⁶etG at similar rates. O⁶iprG was not repaired. Therepair of oligomers containing O⁶meG was only slightly inhibitedby the presence of non-alkylated oligomers. Oligomers containingO⁶etG were only slightly more effective as inhibitors of repairthan the non-alkylated oligomers, indicating that the transferasedoes not bind selectively to alkylated DNA. Parallel structuralstudies have shown that O⁶-alkylguanine:C and O⁴-alkylthymine:Abase pairs have a similar geometry with the alkylated base displacedinto the major groove of the DNA in contrast to O⁶-alkylguanine:Tand O⁴-alkylthymine:G base pairs which retain the Watson-Crickalignment with N1 of the purine juxtaposed to N3 of the pyrimidine.Measurement of the rate of repair of these different base pairssuggests that pairs with the alkyl group exposed in the majorgroove may be repaired more rapidly than those with the alkylgroup more deeply buried in the helix.     

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification

The preparation and characteristics of compound-specific andgroup-specific antibodies against 7-alkylguanines (7-alkGua)are described. A compound-specific antibody against 7-methylguaninewas prepared using a hapten bound to carrier protein throughthe N² position. In a competitive enzyme-linked immunosorbantassay (ELISA) 7-methyl-guanine (7-MeGua) showed 50% inhibition(I_50% at 10 pmol/ well at room temperature, but the inhibitionwas found to be 40 times better at 4°C (I_50% at 250 fmol/well).When the antibody was bound to protein A-Sepharose CL4B 7-MeGuawas retained in immunoaffinity columns. A group-specific antibodyto 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua)bound to carrier protein via the carboxyl group. In a competitiveELISA, this antibody cross- reacted well with 7-CEGua, 7-ethylguanine(7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine(7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinitycolumns prepared from this antibody retained a number of 7-alkGuaof diverse structure. 7-EtGua in calf thymus DNA treated withdiethyl sulphate and ethylnitrosourea was isolated by immunoaffinitypurification and quantified by HPLC-fluorescence. These resultsillustrate the potential of immunoaffinity purification forboth individual DNA adducts and groups of adducts.     

Determination of N-nitrosodimethylamine in artificial gastric juice by gas chromatography-mass spectrometry and by gas chromatography-thermal energy analysis.

The thermal energy analyser (TEA) is considered to be the gold standard for the determination of nitrosamines. However, since many laboratories cannot justify the use of such a very specific detection system, alternative detection methods are useful. While standard gas chromatography (GC) detectors lack the selectivity of the TEA detector, mass spectrometry (MS) seems to be the method of choice to combine GC separation with mass selective detection. Moreover, the detection limits of the GC-MS assay in general use are about 4 times lower than those of the GC-TEA assay. A comparison of GC-MS and GC-TEA data on N-nitrosodimethylamine determinations showed a strong correlation between the two assays (R2 = 0.86), demonstrating the exchangeability of these methods.     

Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation.

A highly sensitive and specific method for the detection of O6-methylguanine (O6-meG), O4-methylthymine (O4-meT), and O4-ethylthymine (O4-etT) has been established by combining prefractionation by high-performance liquid chromatography (HPLC), 32P postlabeling, and immunoprecipitation by monoclonal antibodies (PREPI method). DNA was enzymatically hydrolyzed to 2'-deoxynucleoside-3'-monophosphates (3'-dNps). Each alkyl 3'dNp was separated by reverse-phase HPLC, radiolabeled at the 5' position with [gamma-32P]ATP and polynucleotide kinase. After removing 3'-phosphate for better recognition by the antibodies, the resulting alkyl nucleotides were further fractionated by HPLC and finally precipitated specifically with respective antibodies. The detection limits were 1 fmol for all the alkyl nucleotides analyzed, so that one adduct in 10(8) of its normal counterpart nucleotide can be determined using approximately 100 micrograms (O4-meT and O4-etT) or approximately 150 micrograms (O6-meG) of DNA, i.e., 3-5 x 10(7) cells corresponding to approximately 10 ml of peripheral blood or a few hundred milligrams of tissue. By the use of the PREPI method, three leukocyte and three liver DNA samples from Japanese living in the Tokyo area were analyzed with respect to O-alkyl adduct content. O6-meG was detected in all three of the leukocyte samples (O6-meG:G molar ratio, 1.1 x, 0.8 x, and 1.6 x 10(-8) as molar ratios to guanine). Neither O4-meT nor O4-etT was detected (detection limit, O4-alkylT:thymine molar ratio less than 0.5 x 10(-8)). Among the liver samples analyzed, two cases showed positive O6-meG values (4.2 x and 1.1 x 10(-7) O6-meG:guanine molar ratios). Contrary to the leukocyte DNA, O4-meT (3.9 x, 4.3 x, and 7.5 x 10(-8) as O4-meT:thymine) and O4-etT (1.9 x, 4.9 x, and 8.7 x 10(-8) as O4-etT:thymine) were detected in all the liver samples. These results indicate the validity of the PREPI method for molecular epidemiological studies on DNA alkylation products.     

DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 4-aminobiphenyl are infrequently detected in human mammary tissue by liquid chromatography/tandem mass spectrometry

Some epidemiological investigations have revealed that frequent consumption of well-done cooked meats and tobacco smoking are risk factors for breast cancer in women. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed in well-done cooked meat, and 4-aminobiphenyl (4-ABP) is an aromatic amine that arises in tobacco smoke and occurs as a contaminant in the atmosphere. Both compounds are rodent mammary carcinogens, and putative DNA adducts of PhIP and 4-ABP have been frequently detected, by immunohistochemistry (IHC) or (32)P-post-labeling methods, in mammary tissue of USA women. Because of these findings, PhIP and 4-ABP have been implicated as causal agents of human breast cancer. However, the biomarker data are controversial: both IHC and (32)P-post-labeling are non-selective screening methods and fail to provide confirmatory spectral data. Consequently, the identities of the lesions are equivocal. We employed a specific and sensitive liquid chromatography/mass spectrometry (MS) method, to screen tumor-adjacent normal mammary tissue for DNA adducts of PhIP and 4-ABP. Only 1 of 70 biopsy samples obtained from Minneapolis, Minnesota breast cancer patients contained a PhIP-DNA adduct. The level was three adducts per 10(9) nucleotides, a level that is 100-fold lower than the mean level of PhIP adducts reported by IHC or (32)P-post-labeling methods. The occurrence of 4-ABP-DNA adducts was nil in those same breast tissues. Our findings, derived from a specific mass spectrometry method, signify that PhIP and 4-ABP are not major DNA-damaging agents in mammary tissue of USA women and raise questions about the roles of these chemicals in breast cancer.     

1,N6-Ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabelling

The etheno-bridged exocyclic DNA adducts 1,N⁶-ethenodeoxyadenosine(     

Molecular dosimetry in rat urine of aflatoxin-N7-guanine and other aflatoxin metabolites by multiple monoclonal antibody affinity chromatography and immunoaffinity/high performance liquid chromatography.

The development of molecular dosimetry methods will simplify the identification of people at high risk for cancer. A combined monoclonal antibody immunoaffinity chromatography/high performance liquid chromatography method has been devised to isolate and quantify aflatoxin-DNA adducts and other metabolites in rat urine samples. We report the production of 11 different monoclonal antibodies recognizing aflatoxin B1, aflatoxin Q1, aflatoxin G1, aflatoxicol, and aflatoxin M1 and the application of these antibodies to a multiple monoclonal antibody affinity chromatography technique. Using the multiple monoclonal antibody affinity column with rat urines obtained from dosed animals, between 90 and 95% of total aflatoxin metabolites can be bound to the column and isolated. Analytical immunoaffinity chromatography/high performance liquid chromatography analysis of these isolated aflatoxins reveals that more than 55% of the aflatoxins in rat urine are aflatoxin-dihydrodiol, aflatoxin-N7-guanine, aflatoxin Q1, aflatoxin M1, aflatoxin P1, and aflatoxin B1, accounting for 1.5, 9.6, 1.8, 34.5, 8.0, and 1.0% of the total aflatoxins, respectively. Further, a perchloric acid digestion of the aflatoxin-N7-guanine peak was used to confirm its identity by its conversion to guanine. The measurement of aflatoxin-N7-guanine excretion in rat urine was examined to assess its utility as a marker of DNA adduct formation in the liver, and a dose-dependent excretion in urine was found with a correlation coefficient of 0.99. A comparison of the dose-dependent residual levels of aflatoxin binding to liver DNA with the amount of aflatoxin-N7-guanine excreted in urine showed a correlation coefficient of 0.98. Besides the nucleic acid adduct excretion data, aflatoxin M1 and aflatoxin P1 were evaluated as molecular dosimeters in the urine. Aflatoxin M1 was found to be an excellent marker, whereas no linear relationship between dose and aflatoxin P1 excretion in urine was found.