Proteomics of Bordetella pertussis whole-cell and acellular vaccines
Bordetella pertussis is the etiological agent of whooping cough, a bacterial infection of especially children, which may be fatal without treatment. In frame of studies to investigate putative effects of vaccination on host-pathogen interaction and clonal distribution of strains, in addition to Corynebacterium diphtheriae and Clostridium tetani toxoid vaccines, also whole-cell and acellular pertussis vaccines were analyzed by mass spectrometry.
Sample Processing Protocol
B. pertussis vaccines are typically administered in combination with diphtheria and tetanus toxoid vaccines as DTP3 vaccines. The sample preparation of the vaccines for mass spectrometry analysis was carried out as described recently. The soluble proteins prepared from the vaccine samples were transferred to 10 kDa vivacon 500 membrane filters and the flow-through was discarded after centrifugation. A modified Filter Aided Sample Preparation (FASP) protocol was used for tryptic digest of the prepared vaccines samples. Peptides were collected by centrifugation at 12,000 x g for 20 min. 20 µl of 10 % trifluoroacetic acid (TFA) was added to reach a final concentration of 0.5 % TFA for acidification of the peptide solution. A clean-up of the peptides with C18 stage tips were performed to remove all remaining salts which might interfere with mass spectrometry analyzes. Prior to LC-MS/MS (liquid chromatography tandem-mass spectrometry) analysis, peptides were vacuum dried and solved in 0.1 % trifluoroacetic acid (TFA).
Data Processing Protocol
Mass spectrometric analyses were carried out as described before and resulting raw data files were analyzed using the C. diphtheriae ATCC 700971 / NCTC 13129 / Biotype gravis database (Proteome Id: UP000002198), the Clostridium tetani E88 database, (Proteome Id: UP000001412), and Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) database (Proteome Id.: UP000002676) using the Proteome Discoverer 1.4 program package (Thermo Fisher Scientific, Bremen, Germany). Theoretical masses for peptides were generated by trypsin digestion with a maximum of 2 missed cleavages as described by Schäfer and co-workers. Product ions were compared to the measured spectra using the following parameters: carbamidomethyl modification on cysteine was set as fixed and oxidation of methionine as dynamic modification. Mass tolerance was set to 10 ppm for survey scans and 0.6 Da for fragment mass measurements. For protein identification the thresholds were set on 1 % false discovery rate (FDR).