Background: Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer. Methods: Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis. A targeted multiple reaction monitoring mass spectrometry method was employed to study and quantify cinnamic alcohol along with eight potential phase I or phase II metabolites. The reconstructed human epidermis model, treated with cinnamic alcohol, was also analyzed with a non-targeted high-resolution mass spectrometry method to identify metabolites not included in the targeted method. Results: Two metabolites identified with the targeted method, namely, pOH-cinnamic alcohol and pOH-cinnamic aldehyde, have not previously been identified in a metabolic in vitro system. Their reactivity toward biologically relevant nucleophiles was investigated and compared to their sensitizing potency in vivo in the murine local lymph node assay (LLNA). According to the LLNA, the pOH-cinnamic alcohol is non-sensitizing and pOH-cinnamic aldehyde is a moderate sensitizer. This makes pOH-cinnamic aldehyde less sensitizing than cinnamic aldehyde, which has been found to be a strong sensitizer in the LLNA. This difference in sensitizing potency was supported by the reactivity experiments. Cinnamic sulfate, previously proposed as a potential reactive metabolite of cinnamic alcohol, was not detected in any of the incubations. In addition, experiments examining the reactivity of cinnamic sulfate toward a model peptide revealed no evidence of adduct formation. The only additional metabolite that could be identified with the non-targeted method was a dioxolan derivative. Whether or not this metabolite, or one of its precursors, could contribute to the sensitizing potency of cinnamic alcohol would need further investigation. Discussion: Cinnamic alcohol is one of the most common fragrance allergens and as it is more effective to patch test with the actual sensitizer than with the prohapten itself, it is important to identify metabolites with sensitizing potency. Further, improved knowledge of metabolic transformations occurring in the skin can improve prediction models for safety assessment of skin products.

Propolis is a resin-like material produced by bees fromthe budsof poplar and cone-bearing trees and is used in beehive construction.Propolis is a common additive in various biocosmetics and health-relatedproducts, despite the fact that it is a well-known cause of contactallergy. Caffeic acid and its esters have been the primary suspectsbehind the sensitization potency of propolis-induced contact allergy.However, the chemical structures of the protein adducts formed betweenthese haptens and skin proteins during the process of skin sensitizationremain unknown. In this study, the reactivity of three main contactallergens found in propolis, namely, caffeic acid (CA), caffeic acid1,1-dimethylallyl ester (CAAE), and caffeic acid phenethyl ester (CAPE),was investigated. These compounds were initially subjected to thekinetic direct peptide reactivity assay to categorize the sensitizationpotency of CA, CAAE, and CAPE, but the data obtained was deemed toounreliable to confidently classify their skin sensitization potentialbased on this assay alone. To further investigate the chemistry involvedin generating possible skin allergy-inducing protein adducts, modelpeptide reactions with CA, CAAE, and CAPE were conducted and analyzedvia liquid chromatography-high-resolution mass spectrometry.Reactions between CA, CAAE, and CAPE and a cysteine-containing peptidein the presence of oxygen, both in closed and open systems, were monitoredat specific time points. These studies revealed the formation of twodifferent adducts, one corresponding to thiol addition to the & alpha;,& beta;-unsaturatedcarbonyl region of the caffeic structure and the second correspondingto thiol addition to the catechol, after air oxidation to o-quinone.Observation of these peptide adducts classifies these compounds asprehaptens. Interestingly, no adduct formation was observed when thesame reactions were performed under oxygen-free conditions, highlightingthe importance of air oxidation processes in CA, CAAE, and CAPE adductformation. Additionally, through NMR analysis, we found that thioladdition occurs at the C-2 position in the aromatic ring of the CAderivatives. Our results emphasize the importance of air oxidationin the sensitization potency of propolis and shed light on the chemicalstructures of the resultant haptens which could trigger allergic reactionsin vivo.

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. 

Epoxy resin systems (ERSs) are a class of thermosetting resins that become thermostable and insoluble polymers upon curing. They are widely used as components of protective surfaces, adhesives, and paints and in the manufacturing of composites in the plastics industry. The diglycidyl ether of bisphenol A (DGEBA) is used in 75–90% of ERSs and is thus by far the most used epoxy resin monomer (ERM). Unfortunately, DGEBA is a strong skin sensitizer and it is one of the most common causes of occupational contact dermatitis. Furthermore, DGEBA is synthesized from bisphenol A (BPA), which is a petroleum-derived chemical with endocrine-disruptive properties. In this work, we have used isosorbide, a renewable and nontoxic sugar-based material, as an alternative to BPA in the design of ERMs. Three different bis-epoxide isosorbide derivatives were synthesized: the diglycidyl ether of isosorbide (1) and two novel isosorbide-based bis-epoxides containing either a benzoic ester (2) or a benzyl ether linkage (3). Assessment of the in vivo sensitizing potency of the isosorbide bis-epoxides in the murine local lymph node assay (LLNA) showed that all three compounds were significantly less sensitizing than DGEBA, especially 2 which was nonsensitizing up to 25% w/v. The peptide reactivity showed the same order of reactivity as the LLNA, i.e., 2 being the least reactive, followed by 3 and then 1, which displayed similar peptide reactivity as DGEBA. Skin permeation of 2 and 3 was compared to DGEBA using ex vivo pig skin and static Franz cells. The preliminary investigations of the technical properties of the polymers formed from 1–3 were promising. Although further investigations of the technical properties are needed, all isosorbide bis-epoxides have the potential to be less sensitizing renewable replacements of DGEBA, especially 2 that had the lowest sensitizing potency in vivo as well as the lowest peptide reactivity. 

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.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level. 

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser72, beta Thr84, beta Thr87, beta Ser89, beta His92, beta Cys93, beta Cys112, beta Thr123, and beta His143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with alpha His45, alpha Tyr42, beta Cys93, beta His92, and beta Ser72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser44, beta Thr84, and beta His92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser44, beta Thr84, and beta Ser89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

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.

Glycosylation influences the structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. The types and abundance of glycans on glycoproteins can vary due to diseases such as cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role glycans play in modulating protein function, glycosylation analysis could lead to the discovery of novel biomarkers and is of prime importance in controlling the quality of glycoprotein biopharmaceuticals. Here, we present a method for the identification and quantification of glycoforms directly on intact proteins, after immunoaffinity purification from biological fluids. The method was validated and applied to serum transferrin and the biopharmaceutical trastuzumab. The accuracy of the method, expressed as the relative error (RE), ranged from 2.1 (at high concentrations) to 7.9% (at low concentrations), and intra- and inter-day precision, expressed as relative standard deviation (RSD), was 3.2 and 8.2%, respectively. The sensitivity and linearity of the method were suitable for serum analysis and the LOQ was calculated to be 3.1 and 4.4 mu g mL(-1) for transferrin (TFN) and trastuzumab (TRA), respectively. Its application to transferrin from five healthy human serum samples yielded concentrations between 1.61 and 3.17 mg mL(-1), which are in agreement with blood reference levels. In parallel, the structure of the identified glycans was determined by ion mobility spectrometry coupled with tandem mass spectrometry. No chromatographic separation was required and sample preparation was performed in a semi-automatic manner, facilitating the handling of up to 12 samples at a time. This method should be useful for clinical laboratories and for the quality control of large batches of biopharmaceuticals.

Alteration of glycosylation has been observed in several diseases, such as cancer and neurodegenerative disorders. The study of changes in glycosylation could lead to a better understanding of mechanisms underlying these diseases and to the identification of new biomarkers. In this work the N-linked glycosylation of five target proteins in cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients and healthy controls have been analyzed for the first time. The investigated proteins, transferrin (TFN), alpha-1-antitrypsin (AAT), C1-inhibitor, immunoglobulin G (IgG), and alpha-1-acid glycoprotein (AGP), were selected based on the availability of VHH antibody fragments and their potential involvement in neurodegenerative and inflammation diseases. AD patients showed alterations in the glycosylation of low abundance proteins, such as C1-inhibitor and alpha-1-acid glycoprotein. These alterations would not have been detected if the glycosylation profile of the total CSF had been analyzed, due to the masking effect of the dominant profiles of high abundance glycoproteins, such as IgG. Information obtained from single proteins was not sufficient to correctly classify the two sample groups; however, by using an advanced multivariate technique a total non-error rate of 72 +/- 3% was obtained. In fact, the corresponding model was able to correctly classify 71 +/- 4% of the healthy subjects and 74 +/- 7% of the AD patients. Even if the results were not conclusive for AD, this approach could be extremely useful for diseases in which glycosylation changes are reported to be more extensive, such as several types of cancer and autoimmune diseases.