Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure.

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Verification of Exposure to Novichok Nerve Agents Utilizing a Semitargeted Human Butyrylcholinesterase Nonapeptide Assay.

Novichok (NV) nerve agents were recently added to the list of Schedule 1 chemicals of the Chemical Weapons Convention. There is a well-accepted method for assessment of nerve agent exposure based on mass spectrometric analysis of a nonapeptide with the serine-198 residue modified by the nerve agent, but this approach has not yet been reported for the class of NV agents and requires the availability of reference standards, which may be a limitation for NV agent exposure assessment. Thus, a goal of this study was to first verify the utility of the nonapeptide method for the characterization of human plasma samples exposed in vitro to the NV agents A-230, A-232, and A-234. A second aim was to evaluate the possibility of identifying unknown exposures by applying precursor ion scanning in combination with high resolution mass spectrometry (HRMS). Thus, precursor ion scanning, with a generic fragment ion (m/z 778) of the nonapeptide, was used to pinpoint any modified nonapeptide, while HRMS was used for structural elucidation of the adduct moiety. By this approach, use of HRMS enabled differentiation between adducts of agents with similar molecular masses. A new unique feature that could be exploited for NV nonapeptide analysis was that the modification was released from the peptide during fragmentation in the mass spectrometer and was detected in the low-mass region of the mass spectrum. This low-mass region was extremely informative and contributed to the assignment of the structure of the particular agent used, which is especially important in case no reference materials are available. The presented method is important for verification purposes by the Organisation for Prohibition of Chemical Weapons (OPCW), e.g., in case of investigations of alleged use of NV agents, and for regular forensic investigations.

Mass spectrometric analysis of adducts of sulfur mustard analogues to human plasma proteins: approach towards chemical provenancing in biomedical samples.

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

Mass spectrometric identification of isocyanate-induced modifications of keratins in human skin.

In the current paper we show that exposure of human callus to isocyanates leads to covalent modifications within keratin proteins. Mass spectrometric analyses of pronase digests of keratin isolated from exposed callus show that both mono- and di-adducts (for di-isocyanates) are predominantly formed on the ε-amino group of lysine. In addition, numerous modified tryptic keratin fragments were identified, demonstrating rather random lysine modification. Interestingly, preliminary experiments demonstrate that in case of MDI a similar lysine di-adduct was formed with lung elastin. Our data support the hypothesis that skin sensitization through antigenic modifications of skin proteins by isocyanates could play a role in occupational isocyanate-induced asthma. It is further envisaged that the elucidated adducts will also have great potential for use as biomarkers to assess skin exposure to isocyanates. Advantageously, the various lysine adducts display the presence of a characteristic daughter fragment at m/z 173.1 [lysine-NCO](+), enabling generic and rapid screening for exposure to isocyanates.