Characterization of adducts formed between human serum albumin and the butadiene metabolite epoxybutanediol

Abstract

1,3-Butadiene (BD) has been classified as a potential human carcinogen. It occurs in the environment as well as in industrial settings. In humans, BD is readily metabolized to reactive epoxides (e.g. 1,2-epoxy-3,4-butanediol). In this study, conjugates between human serum albumin (HSA) and EBD were synthesized (molar ratios of 1:600, 1:1 and 1:0.1; HSA/EBD) under physiological conditions. The 1:600 conjugate and a blank HSA sample were digested with trypsin to obtain specific peptides that were fractionated by preparative liquid chromatography (LC). The fractions were analyzed using nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoES-QqTOFMS). Adducted HSA tryptic peptides were identified and the adducted amino acid residues were identified by sequence analysis based on tandem mass spectrometry (MS/MS). A total of 26 2,3,4-trihydroxybutyl (THB) adducts were identified on 23 tryptic peptides in the HSA/EBD conjugate. The adducted amino acids were the N-terminal aspartic acid residue, six glutamic acid residues, five histidine residues and 14 lysine residues. Results from the nanoES-QqTOFMS experiments were used to set up a more sensitive liquid chromatographic/mass spectrometric (LC/MS) analysis using selected reaction monitoring. Eight of the adducted peptides could be detected in tryptic digests of the 1:0.1 HSA/EBD conjugate.

Mass spectrometric characterization of covalent modification of human serum albumin by 4‐hydroxy‐trans‐2‐nonenal

“…Since Lys525 has been reported to be an important adduct‐binding site of HSA,28 consumption of peptide T49 (QTALVELVK) was first explained by considering the formation of the merged peptide KQTALVELVK as a consequence of the covalent adduction to Lys525, and the multicharged ions relative to the corresponding MA and SB HNE adducts were predicted. The reconstituted ion traces evidence the formation of both the adducts (Fig.…”

“…Freshly isolated HSA is heterogeneous with respect to its thiol content, reported as typically 0.6–0.7 SH/HSA, as confirmed in the present study. Another important and reactive binding site of HSA toward HNE is represented by His146, a nucleophilic site that has been previously reported to be the adduction site of polycyclic aromatic hydrocarbon epoxides32 and of the butadiene metabolite epoxybutanediol 28. Lys199 has been reported as a reactive nucleophilic site located in the IIA binding site of albumin, capable of reacting with several ligands such as benzylpenicillin, through an aminolysis reaction 33.…”

Strategy for identification and detection of multiple oxidative modifications within proteins applied on persulfate‐oxidized hemoglobin and human serum albumin

“…The software packages generated a long list of peptides differing from the control sample that was manually verified. This verification process was a rather time‐consuming process, but in previous work even more time‐consuming strategies have been used such as acid hydrolysis of different fractions or manual comparison 14, 16, 17. Thus, the usage of these software packages markedly shortened the analysis time.…”

Mass Spectral Analyses of Hydroxymethylvinyl Ketone-Hemoglobin Adducts Formed after in Vivo Exposure of Sprague−Dawley Rats to 3-Butene-1,2-diol

“…Multiple adducts on globin chains and multiple internal peptides indicate that sites of modification other than N-terminal valine may also be involved. THB adducts were previously identified with N-terminal aspartic acid, glutamic acid, histidine, and lysine residues of albumin in vitro when human serum albumin was incubated with EBD at physiological conditions (29). …”