Structural elucidation of isocyanate-peptide adducts using tandem mass spectrometry

Abstract

Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when isocyanates are reacted with model peptides, tandem mass spectrometry was employed to unambiguously identify the binding site of four commercially-relevant isocyanates on model peptides. In each case, the isocyanates react preferentially with the N-terminus of the peptide. No evidence of side-chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking was observed between the N-terminal amine and a side-chain amine of arginine, when Arg was located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization of the isocyanate at the N-terminus, rather than via addition of multiple isocyanate molecules to varied residues within the peptide. The direct observation of isocyanate binding to the N-terminus of peptides under these experimental conditions is in good agreement with previous studies on the relative reaction rate of isocyanate with amino acid functional groups.

Mass spectrometric identification of isocyanate-induced modifications of keratins in human skin

“…In addition to the e-amino lysine adducts, we identified several other isocyanate-modified amino acids, in line with the results published by Hettick et al [21] and Sabbioni et al [22,23]. These modifications are most likely the result of isocyanate binding to the a-amino group of N-terminal amino acids of the various keratins and constituted an almost negligible fraction of the total modified residues.…”

“…Although evidence for conjugation of isocyanates with keratins was already presented by Wisnewski et al [25] and more recently also by Nayak et al [26], no mass spectrometry data of discrete adducts were reported in these studies. Both mono-and di-adducts are predominantly formed on the e-amino group of lysine residues, in line with results published for other proteins [19,21]. Keratins have highly homologous amino acid sequences and are also abundantly present in the lung epithelium.…”

Monoclonal Antibodies Against Toluene Diisocyanate Haptenated Proteins from Vapor-Exposed Mice

“…We recently demonstrated the potential for such methods for isocyanatepeptide adducts using tandem mass spectrometry. (23) The MAbs may also be useful reagents for the development of immunoassays designed for dNCO exposure assessments.…”

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition

“…LC-MS/MS analysis was performed as previously described (17, 18) . Briefly, samples were brought to 45 mM ammonium bicarbonate, reduced with tributylphosphine (TBP), and alkylated with iodoacetamide (IAA) according to manufacturer’s instructions (Pierce).…”