Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.

We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.