Characterization of pertussis toxoid by two-dimensional liquid chromatography–tandem mass spectrometry

Pertussis toxoid, an acellular pertussis vaccine prepared by hydrogen peroxide treatment in the presence of Fe3+, has not been well characterized. Because the toxoid has been a part of the DTaP vaccine for infants, it is of interest and significance to have a clear understanding of its structure. The five subunits of pertussis toxin (PT) have a combined molecular weight of approximately 95,000 Da. The peroxide treatment in toxoid formation introduces additional complexity into the protein sequence. To maximize sequence coverage, a two-dimensional liquid chromatography–tandem mass spectrometry (2D LC–MS/MS) approach was used to analyze the tryptic digest of toxoid as a whole. An analytical-scale high-performance liquid chromatography (HPLC) instrument using a pentafluorophenyl (PFP) column was used as the first-dimensional LC for fraction collection. The fractions were then analyzed by nanoLC–MS/MS using a C18 column to acquire collision-activated dissociation (CAD) spectra of the tryptic peptides. It is shown that a PFP column has a different peptide retention specificity from a C18 column. A combination of a PFP column and a C18 column is a viable approach for dispersing peptides in a complex mixture. From the structures of 65 peptides that represented approximately 50% of its sequence, PT was found to have sustained heavy oxidative damages during toxoid preparation. Nearly all methionine, cysteine, and (likely) tryptophan residues were oxidized. Evidence of histidine and tyrosine oxidation was also observed. In addition, a large percentage of asparagine was found hydrolyzed to aspartic acid. These findings corrrelate well with the reduction of PT toxicity by peroxide treatment.