Development of a targeted nanoLC-MS/MS method for quantitation of residual toxins from Bordetella pertussis

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis) infection. Pertussis pathogenesis is driven by cell-surface adhesion proteins and secreted toxins; some of which have been harnessed for their immunogenic properties as purified antigen components in acellular vaccines. Two of these virulence factors, adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), are protein toxins with potential for co-purification, and therefore must be monitored as process-related impurities during the development of acellular Pertussis vaccine candidates. Here we describe the development of a targeted nanoLC-MS/MS method for absolute quantitation of ACT and DNT in process intermediates from acellular Pertussis antigen purification. Starting from an in silico digest of the toxin sequences, a synthetic peptide screening approach was applied to systematically evaluate candidate sequences as surrogates for protein quantitation. Following refinement to a subset of sequences, absolutely quantified heavy-labelled (AQUA) peptides were implemented in a parallel reaction monitoring (PRM) workflow with limits of detection (LOD) and quantitation (LOQ) in the 12.5-25 amol (2-4 ng/mL) range on-column. In this work, we highlight a 'standards-driven' approach to surrogate peptide selection for protein quantitation. This strategy can be broadly applied in the absence of purified reference material and accelerate quantitative LC-MS method development across multiple sample matrices.