Application of Proteomic Workflows for the Identification of Biomarkers for the Retrospective Verification of Sulfur Mustard Intoxication

The family of sulfur mustards, as potent alkylating agents, has been used as chemical warfare agents for over a century. The retrospective verification of exposure and, hence, the confirmation of alleged use of such agents, remains a challenging task with high demand for resources in the identification, preparation, and analysis of potential specific biomarkers. We report a facile, high-throughput methodology appropriate for biomedical samples using proteomic workflows and the implementation of statistical models. Following the strategy, full proteome analysis of blood serum exposed to three different sulfur mustard representatives, namely, mustard gas, sesquimustard, and O-mustard, revealed 270 potential biomarkers with 213 novel alkylation sites. Targeted sample preparation by human serum albumin enrichment using Cibacron Blue-modified magnetic beads and analysis in prm-PASEF mode allowed the unequivocal retrospective verification of in vitro exposure to a level of 5 μM final concentration of agent in serum. Finally, the synthesis of four alkylated amino acid building blocks (Glu, Asp, Cys, and His) and their implementation in four different representative peptide sequences are demonstrated.