Abstract
Aims: We present the discovery of functional yersiniophage and Yersinia pestis (Y. pestis) peptide targets for monitoring bacteriophagebased amplification processes and species-specific identification of Yersinia pestis, the etiological agent of plague. Methods: For protein confirmation and peptide target discovery, ultra performance liquid chromatography and hybrid tandem mass spectrometry was utilized to identify proteolytic cleaved peptides generated from digestion of whole phage and bacterial lysates. Peptide discovery was accomplished with a UPLC NanoAcquity interfaced into a Q-TOF Premier (Waters Corporation) for full scan and MS/MS experiments. Targeted development utilized the MRM initiated detection and sequencing (MIDAS) workflow design software (Applied Biosystems) to create in silico MRM transitions which optimized collision energies for tryps in cleaved peptides from the proteins of interest. The analytical column utilized was an UPLC bridged ethyl hybrid (BEH) 1.0 × 50 mm reverse phase C18 (1.7-μim particle size) interfaced into a 4000 Q TRAP for MRM triggered MSMS. Results: We screened yersiniophage phiA1122, R, V, and Y proteins for conserved peptide targets for utilization in routine mass spectrometry monitoring of the phage amplification process. We also identified murine toxin and F1 antigen peptide targets that are unique to Yersinia pestis, which upon lysis, can be utilized for confirmatory identification of Y. pestis. Conclusions: Major and minor capsid protein peptides will serve to enhance quantifiable metric measurements of the amplification process for rapid yersiniophage diagnostic capabilities and concomitant determination of the presence of viable Y. pestis bacilli. Moreover, the targeted murine toxin peptide directly confirmed the presence of Y. pestis at 26°C and can be applied as an additional tool for zoonosis surveillance capabilities in a clinical laboratory setting. Significance and Impact of Study: Applications utilizing these peptide targets identified in this work may be beneficial in emergency response situations requiring presumptive as well as confirmatory identification of Y. pestis bacteria.