Protein adducts analysis

This paper will focus on methods that are based on the analysis of long-lived protein adducts of CW agents which are detectable weeks or even months after exposure. Examples of real exposure incidents will be described. 

In the case of sulfur mustard, analysis of low molecular weight urinary metabolites suffers from the same drawback as in the case of anticholinesterases, i.e., these products are rapidly excreted and provide therefore limited retrospectivity. Similarly, the in vivo lifetime of DNA adducts of sulfur mustard are less than those of protein adducts due to repair of DNA damage. 

Covalent protein adducts of quinones are formed through Mchael-type addihon reachon with protein sulfhydryl groups or glutathione. Metabolic activahon of several toxins (e.g., naphthalene, pentachlorophenol, and benzene) into quinones has been shown to result in protein quinone adducts (Lin et al, 1997 Rappaport et al, 1996 Zheng et al., 1997). Conversion of substituted hydroquinones such as p-aminophenol-hydroquinone and 2-bromo-hydroquinone to their respective glutathione S-conjugates must occur to allow bioactivation into nephrotoxic metabolites (Dekant, 1993). Western blot analysis of proteins from the kidneys of rats treated with 2-bromo-hydroquinone has revealed three distinct protein adducts conjugated to quinone-thioethers (Kleiner et al, 1998). 

M. Tornqvist, C. Fred, J. Haglund, H. Helleberg, B. Paulsson and P. Rydberg, Protein adducts quantitative and qualitative aspects of their formation, analysis and applications, J. Chromatogr., B, 778, 279-308 (2002). 

Carol-Visser, J., Van der Schans, M., Fidder, A., Hulst, A.G., Van Baar, B.L.M., Irth, H., Noort, D. (2008). Development of an automated on-line pepsin digestion-liquid chromatography-tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents. J. Chromatogr. B 870 91-7. 

Holland, K.E., Solano, M.I., Johnson, R.C., Maggio, V.L., Barr, J.R. (2008). Modifications to the organophosphorus nerve agent-protein adduct refluoridation method for retrospective analysis of nerve agent exposures. J. Anal. Toxicol. 32(1) 116-24. 

Boysen G and Hecht SS (2003) Analysis of DNA and protein adducts of benzo [a]pyrene in human tissues using structure-specific methods. Mutation Research 543 17-30. 

In the case of the ESTMS-MS data, actual amino acid sequence can be deduced. This is possible due to the CID processes, which breaks the peptides further into amino acid ions. Each amino acid ion has a specific mass and by calculating masses of specific amino acid from the MS spectra, the exact sequence of the peptide and in turn the protein can be deduced. The workhorse of such analysis is a program called Sequest . Since the ESTMS-MS analysis provides information about the actual amino acid sequence, it is also useful to obtain information about protein modifications (such as phosphorylation) and toxicant-induced protein adducts. This has become even easier with the advent of new software tools and highly intelligent algorithms such as SALSA . 

Korte WD, Walker EM, Smith JR, et al. The determination of sulfur mustard exposure by analysis of blood protein adducts. Wehrmed. Mschr, 2005 49 327. 

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