Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin

Abstract

An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a ‘housekeeping peptide’, and annotates modifications based on a combination of retention time, accurate mass, elemental composition and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g. ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.

Using lysine adducts of human serum albumin to investigate the disposition of exogenous formaldehyde in human blood

“…This method is analogous to that used by Grigoryan et al, who quantified HSA adducts, modified at Cys34, in tryptic digests relative to a ‘housekeeping peptide’, which was also derived from HSA. Using synthetic standards, the authors showed that the peak area ratios (Cys34 adduct/housekeeping peptide) were highly linear over a wide dynamic range of adduct concentrations (Grigoryan et al, 2016). …”

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

“…The elucidation of the binding sites of xenobiotics to proteins is useful for the elucidation of the potential immunogenic properties of the adduct 92 or to develop strategies to measure multiple adducts after isolating a peptide of Alb with a nucleophilic hot-spot such as Cys34. 6,93,94 …”

“…Recently, a rapid method was developed to obtain about 0.1 mg of Alb with a 72–75% purity. 94 Plasma (5 μL) was added to 60 μL of 50% methanol and incubated at room temperature for 15 min with constant agitation. The samples were centrifuged to remove precipitates and immediately diluted with four volumes of digestion buffer, and digested with trypsin.…”

“…The samples were centrifuged to remove precipitates and immediately diluted with four volumes of digestion buffer, and digested with trypsin. 94 …”

“…93,94,154 The screening technique detected a presumed adduct of acrylonitrile, but it failed to detect T3 adducts of other prominent carcinogens formed in tobacco smoke such as acrylamide, benzene and butadiene, among others. 94 Another enrichment approach has been to design a polyclonal anti-T3 antibody of Alb that could be employed to capture Cys34 adducts in tryptic digests of Alb. 317 These enrichment methods and bioanalytical approaches are promising, but they require further improvements in LC-MS analyses and more comprehensive scanning methods, such as DIA, particularly for those adducts formed at low abundance.…”