Technical Abstract: We have identified the protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five different strains of Campylobacter upsaliensis and one strain of C. helveticus by proteomic techniques. Only one of these strains (C. upsaliensis strain RM3195) had previously been genomically sequenced. The significant findings are as follows. 1. The protein biomarkers identified were: 10 kD chaperonin, protein of unknown function (DUF465), phnA protein, probable periplasmic protein, D-methionine-binding lipoprotein MetQ, cytochrome c family protein, DNA-binding protein HU, thioredoxin, asparigenase family protein, helix-turn-helix domain protein as well as several conserved hypothetical and ribosomal proteins. 2. Amino acid substitutions in protein biomarkers between species and strains account for variations in biomarker ion mass-to-charge (m/z). 3. The most common post-translational modifications (PTMs) were cleavage of N-terminal methionine and signal peptides. The rule that predicts N-terminal methionine cleavage, based on the penultimate residue, does not appear to apply to C. upsaliensis proteins when the penultimate residue is threonine. 4. It was discovered that the genes of several of the protein biomarkers of sequenced strain were found to have nucleotide sequences with gtg or ctg or ttg "start" codons which were not the actual start codon of the protein based on proteomic analysis. 5. Proteomic identification of the protein biomarkers of the other C. upsaliensis and C. helveticus strains involved identification of homologous protein amino acid sequences to that of the sequenced strain. Interestingly, some protein sequence regions that were not completely homologous to the sequenced strain, due to amino acid substitutions, were found to have homologous sequence regions from more phyogenetically distant strains of Campylobacter, e.g. C. jejuni, C. coli, etc. Exploiting this partial homology of more distant species/strains, it was possible to construct a composite amino acid sequence using multiple partial homologous sequence regions from both phylogenetically proximate and distant strains. The new composite sequence was confirmed by both MS and MS/MS data. Thus, it was possible to determine the amino acid sequence of an unknown protein biomarker from a genomically un-sequenced bacterial strain without the necessity of either genetically sequencing the biomarker gene or resorting to the more challenging de novo MS/MS sequencing.