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Your Environment. Your Health.

Progress Reports: Massachusetts Institute of Technology: Proteins and DNA - New Methods of Adduct Detection

Superfund Research Program

Proteins and DNA - New Methods of Adduct Detection

Project Leader: Steven R. Tannenbaum
Grant Number: P42ES004675
Funding Period: 1995 - 2000

Progress Reports

Year:   1998  1997  1996  1995 

The feasibility of using nanoelectrospray-tandem mass spectrometry to determine PAH-albumin adducts without prior characterization was evaluated. Nanoelectrospray (nanoES) is a technique pioneered by others that utilizes small sample volumes and extremely low flow rates (ca. 20-40 nL/min). The resulting long duration of the analysis permits multiple MS/MS experiments to be performed with a single sample. Whole enzyme digests of HSA adducted with 5-methylchrysene or benzo[g]chrysene were analyzed by nanoES. Precursor ion scans were performed with Q3 of a triple-stage quadrupole mass spectrometer set to detect ions characteristic for the hydrocarbon moiety. Molecular ions detected in this way were then subjected to product ion analysis to provide spectra that consisted predominantly of ions corresponding to the hydrocarbon moiety and a peptide. The latter was sequenced by changing the MS operating parameters to induce in-source decomposition, followed by conventional tandem MS analysis of the peptide ion. This process could, in principle, be used to search for unknown hydrocarbon adducts by starting with precursor ion analysis with Q3 set to detect ions characteristic of a selected peptide such as the well-defined subsequence that includes His(146) and is produced when adducted albumin is digested with proteinase K.

Research has focused primarily on improvements to the ADAM procedure (adduct detection by acylation with methionine) for post-labeling of DNA adducts. This procedure is characterized by chemical, rather than enzymatic, introduction of a radiolabel into a DNA adduct after its isolation. As research progressed, it became apparent that fluorescent dyes had the potential to offer considerably more sensitivity than could be achieved with radioactive 35S from methionine. Thus, the basic approach developed for ADAM was extended to include acylation with fluorescent reagents.

The fluorescent dye, BODIPY FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) was used to label deoxynucleosides including deoxyguanosine (dG), O6-methyl-dG, and 8-hydroxy-dG. Initial experiments with dG showed that dye to dG ratios greater than 10 produced a single product, the bis-BODIPY-labeled dG, within 2 hours at ambient temperature and the yield exceeded 90%. In general, the labeling reaction was carried out at 25 oC for 2 hours in anhydrous dimethyl formamide with 20-fold excess of BODIPY FL and the activating agents, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine. Under these conditions, all of the adducts tested showed quantitative conversion to bis-labeled products, as confirmed by electrospray MS. Labeling of a set of protected dG with protecting groups at different positions indicated that BODIPY derivatives formed by reaction with the 3'- and 5'-hydroxyl groups. Detection limits at present are in the range of 100 amol. Accurate quantitation is accomplished by using as internal standard the same adduct labeled with a homologous BODIPY dye, which can be separated chromatographically.

Research has also focused on making improvements to the previously reported HPLC-LIF method for quantitative analysis of albumin adducts of benzo[a]pyrene diol epoxide (BPDE) in order to achieve increased sensitivity and reduce interferences. The need for improvement became apparent during the prior funding period when it was observed from analysis of 100 serum specimens that the positivity rate was less than 20%. Attention was focused primarily on several aspects of sample preparation, with particular attention to monoclonal antibody affinity purification of the adduct following its release from the protein by enzymatic digestion. A microscale immunoaffinity chromatography system was developed that requires only 20 g of antibody per sample in custom-fabricated low volume columns. The reduced solvent volume required, together with the elimination of contact with plastics has largely eliminated the impurities that led to interfering signals. Preliminary results indicate that the positivity rate observed in the analysis of randomly selected serum specimens is nearly 100%.

For AMS to fulfill its promise as an ultrasensitive isotope detector, it will be necessary to devise interfaces that accept the output of chromatographs and related separation systems, convert their analytes to a form that is suitable for the AMS ion source, and do so with little loss of peak shape and resolution. Although not designed specifically for AMS, prototypes of GC interfaces have already been described. Project researchers, in collaboration with Newton Scientific, Inc., have adapted the CuO-based reactor approach to the specific requirements of a low-energy AMS.

The reactor is modeled after published designs except that it is connected directly to the ion source. For the experiments described here, the ion source was part of a conventional mass spectrometer used to monitor CO2 produced by the reactor. Direct connection to the ion source resulted in low pressure in the reactor and in this way it was possible to preserve GC peak shape exceptionally well. Measuring the conversion of methane to CO2, since methane is known to be far more difficult to oxidize than most other organic compounds, tested the efficiency of the reactor. Yields were at least 97% by comparison with a CO2 standard. Mixtures of organic compounds including sulfur- and chlorine-containing compounds were separated by GC and passed through the reactor. Resulting peaks of CO2 were comparable in area, with variation of ±10%. Such variation could easily arise from differences in chromatographic performance. Thus, oxidation of different carbon atoms within the same molecule was tested with a series of compounds specifically labeled with 13C. Within the measurement error (±1%), no differences could be observed. Project researchers are now satisfied that the reactor/interface design is suitable for the purpose, have transferred the equipment to the AMS low energy analyzer, and have begun experiments with that arrangement.

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