An Adductomic Approach to Identify Electrophiles In Vivo

Abstract

Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA, constituting a risk for toxic effects. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This Mini-Review focuses on the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in haemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC-MS/MS. In the first application of the approach, samples from 12 smokers/ non-smokers were screened for Hb adducts, and six previously identified adducts and 20 unknown adducts were detected. To confirm the observation of the detected unknown adducts, targeted screenings were performed in larger sets of blood samples (n = 50-120) from human cohorts. The majority of the previously detected unknown adducts was found in all analysed samples, with large interindividual variations in adduct levels. For structural identification of unknown adducts, a strategy using adductome LC-MS/MS data was formulated and applied. Six identified adducts correspond to ethylation and the precursor electrophiles ethyl vinyl ketone, glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. The observation of these adducts in human blood motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.

Mode of action-based risk assessment of genotoxic carcinogens

“…Carlsson et al (2014) presented a novel screening strategy for unknown Hb adducts using the modified Edman degradation with Fluorescein-5-isothiocyanate (FITC) as the cleavage reagent for the N-terminal Val. The technique allowed detecting seven known and 19 unknown Val adducts in human Hb (Carlsson and Tornqvist 2017;Carlsson et al 2014).…”

Discovery of Novel N-(4-Hydroxybenzyl)valine Hemoglobin Adducts in Human Blood

“…13,15,16 The most abundant N-terminal Hb adduct in human blood measured was the previously identified methyl modification, followed by carboxymethylation. 17 High levels of protein methylation in human blood can be explained by the endogenous methylating agent S -adenosylmethionine. 18 The carboxymethyl adducts are formed as a result of degradation of glycated Hb (HbA1c), 19,20 or from reaction with glyoxal, which comes mostly from dietary sources.…”

A simplified method for detection of N‐terminal valine adducts in patients receiving treosulfan

“…Importantly, these results show that this simplified method can quantitate environmental background and treosulfan‐induced pyr ‐Val and THB‐Val N ‐terminal valine globin adducts in humans. Utilization of the Poroshell column technology enables adduct analysis without the need for adduct enrichment such as the FI R E assay, immunoaffinity purification or modified Edman degradation and gas chromatography . The simplicity of the applied method suggests that it can be expanded to unbiased adduct profiling, using the next generation of more sensitive and specific mass spectrometers …”

“…Utilization of the Poroshell column technology enables adduct analysis without the need for adduct enrichment such as the FIRE assay, immunoaffinity purification or modified Edman degradation and gas chromatography. 16,19,[28][29][30][31][32] The simplicity of the applied method suggests that it can be expanded to unbiased adduct profiling, using the next generation of more sensitive and specific mass spectrometers. [33][34][35]…”