Skin (contact) allergy, the most predominant form of immunotoxicity in humans, is caused by small electrophilic compounds (haptens) that modify endogenous proteins. Approximately 20% of the general population in the Western world is affected by contact allergy. Although the importance of the hapten-protein conjugates is well established in the initiation of the immunological reaction, not much progress has been made regarding identification of these conjugates in vivo or exploration of their potential as diagnostic tools. In this study, the human serum albumin (HSA) and human hemoglobin (Hb) adductome for three representative contact allergens with different chemical properties, 1-chloro-2,4-dinitrobenzene (DNCB), 1,2-epoxy-3-phenoxypropane (PGE), and 2-bromo-2-(bromomethyl)glutaronitrile (MDBGN), were studied. Plasma and red blood cell lysate were used as a source for HSA and Hb, respectively. The Direct Peptide Reactivity Assay was used to investigate adduct formation of MDBGN with nucleophilic moieties and revealed that MDGBN is converted to 2-methylenepentanedinitrile in the presence of sulfhydryl groups prior to adduct formation. Following incubation of HSA and Hb with haptens, an Orbitrap Q Exactive high-resolution mass spectrometer was used to perform an initial untargeted analysis to screen for adduct formation, followed by confirmation by targeted Parallel Reaction Monitoring analysis. Although a subset of adducted sites was confirmed by targeted analysis, only some of the adducted peptides showed an increase in the relative amount of the adducted peptide with an increased concentration of hapten. In total, seven adduct sites for HSA and eight for Hb were confirmed for DNCB and PGE. These sites are believed to be the most reactive. Further, three of the HSA sites (Cys34, Cys62, and Lys190) and six of the Hb sites (subunit α: Val1, His45, His72; subunit β: Cys93, His97, and Cys112) were haptenated already at the lowest level of hapten to protein molar ratio (0.1:1), indicating that these sites are the most likely to be modified in vivo. To the best of our knowledge, this is the first time that the adductome of Hb has been studied in the context of contact allergens. Identification of the most reactive sites of abundant proteins, such as HSA and Hb, is the first step toward identification of contact allergy biomarkers that can be used for biomonitoring and to develop better diagnostic tools based on a blood sample.
|Original language||English (US)|
|Number of pages||14|
|Journal||Chemical research in toxicology|
|State||Published - Oct 19 2020|
Bibliographical noteFunding Information:
This project was supported by the Swedish Research Council for Sustainable Development (FORMAS 2017-01511), Birgit and Hellmuth Hertz' Foundation (Royal Physiographic Society of Lund), and Stockholm University. L.N.E. and N.Y.T. were partially supported by grants from the US National Institutes of Health (CA095039, ES023350) and the University of Minnesota Twin Cities.
This project was supported by the Swedish Research Council for Sustainable Development (FORMAS 2017-01511), Birgit and Hellmuth Hertz’ Foundation (Royal Physiographic Society of Lund), and Stockholm University. L.N.E. and N.Y.T. were partially supported by grants from the US National Institutes of Health (CA095039, ES023350) and the University of Minnesota, Twin Cities.
© 2020 American Chemical Society.