Methionine³²⁹ in human serum albumin: A novel target for alkylation by sulfur mustard

Abstract

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering.Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine 329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion.The alkylated tetrapeptide LeuGlyMet 329 (-HETE)Phe, LGM(-HETE)F, was detected after pepsin-mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry.Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum).Stability of the alkylated Met 329 in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure

“…While secondary markers are not unambiguous for identification but able to support identifications based on primary ones, data has to be reported from at least one primary biomarker. For the identification of SM in human plasma samples, the primary biomarkers are HETE-CP (from HSA), HETE-CPF (from HSA), N1/N3-HETE-His (from HSA and Globin), and other protein-or amino acid-adducts (e.g., AE 230 (-HETE) VSKL from HSA [12] and LGM 329 (-HETE) F from HSA, [9]), while the secondary marker for SM is thiodiglycol [41]. Our aim was to develop methods for the analysis and identification of HETETE-CP and HETETE-CPF as primary biomarkers of Q in plasma.…”

“…Q and SM competed for the thiol groups of a limited amount of Cys 34 applied as neat HSA instead of plasma to minimize any side reactions with other plasma proteins. As numerous amino acids of HSA are expected to be prone to alkylation as exemplarily shown for Glu 230 [12] and Met 329 [9], a low HSA concentration was applied. After incubation, samples were subjected to pronase-mediated proteolysis to monitor HETE-CP and HETETE-CP simultaneously as measures of their respective HSA-adducts.…”

“…The most long-lasting in vivo biomarkers of exposure are protein-adducts such as those formed with human serum albumin (HSA). HSA-adducts result from covalent linkage of SM to, e.g., the Cys 34 residue, and have emerged as the most prominent biomarkers [7][8][9][10]. They have been used on several occasions for the analysis of samples from OPCW interlaboratory exercises but also from exposed victims [11][12][13][14].…”

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

“…After alkylation, a Met sulfonium ion is formed containing a permanent positive charge at the sulfur atom (Kramer and Deming 2012) which may cause conformational changes in the protein tertiary structure or may even cause disturbance of the protein secondary structure (Kramer and Deming 2013). Recently, it was shown that Met residues may also be alkylated by SM as proven for Met 329 in human serum albumin (Siegert et al 2019).…”

“…Treatment of CK with the alkylating chemical warfare agent sulfur mustard (SM) resulted in the formation of the specific hydroxyethylthioethyl-(HETE)-moiety at Cys 283 (Lüling et al 2021;Steinritz et al 2021) but its effect on enzyme activity has not been addressed so far. This was investigated in the present study with particular focus on Cys 283 but also on reactive Met residues, that in principle might also be essential for the activity of certain enzymes (Rogers et al 1976) and were shown to be a potential target of alkylation by SM (Siegert et al 2019).…”