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 (Cys(34), Cys(62), and Lys(190)) and six of the Hb sites (subunit a: Val(1), His(45), His(72); subunit beta: Cys(93), His(97), and Cys(112)) 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.

Hemoglobin (Hb) adducts are widely used in human biomonitoring due to the high abundance of hemoglobin in human blood, its reactivity toward electrophiles, and adducted protein stability for up to 120 days. In the present paper, we compared three methods of analysis of hemoglobin adducts: mass spectrometry of derivatized N-terminal Val adducts, mass spectrometry of N-terminal adducted hemoglobin peptides, and limited proteolysis mass spectrometry . Blood from human donors was incubated with a selection of contact allergens and other electrophiles, after which hemoglobin was isolated and subjected to three analysis methods. We found that the FIRE method was able to detect and reliably quantify N-terminal adducts of acrylamide, acrylic acid, glycidic acid, and 2,3-epoxypropyl phenyl ether (PGE), but it was less efficient for 2-methyleneglutaronitrile (2-MGN) and failed to detect 1-chloro-2,4-dinitrobenzene (DNCB). By contrast, bottom-up proteomics was able to determine the presence of adducts from all six electrophiles at both the N-terminus and reactive hemoglobin side chains. Limited proteolysis mass spectrometry, studied for four contact allergens (three electrophiles and a metal salt), was able to determine the presence of covalent hemoglobin adducts with one of the three electrophiles (DNCB) and coordination complexation with the nickel salt. Together, these approaches represent complementary tools in the study of the hemoglobin adductome. 

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.