For the verification of exposure to the banned
blister agent
sulfur mustard (SM) and the better understanding of its pathophysiology,
protein adducts formed with endogenous
proteins represent an important field of toxicological research. SM and its analogue
2-chloroethyl ethyl sulfide (
CEES) are well known to alkylate nucleophilic
amino acid side chains, for example, free-
thiol groups of
cysteine residues. The specific two-dimensional
thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of
maleimide dyes allows the staining of free
cysteine residues in
proteins. As a consequence of alkylation by, for example, SM or
CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with
CEES and subsequent matrix-assisted
laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed
transthyretin (TTR) as a target of
alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-
HETE)PLMVK was alkylated by the hydroxyethylthioethyl (
HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-
HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary
protein marker molecule for the verification of SM exposure.