Software for top-down proteomic identification of a plasmid-borne factor (and other proteins) from genomically sequenced pathogenic bacteria using MALDI-TOF-TOF-MS/MS and post-source decay

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A plasmid-borne factor (hypothetical protein) was identified in three Shiga toxin-producing Escherichia coli O113:H21 strains using antibiotic-induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS), post-source decay (PSD) and top-down proteomic analysis. The plasmid-encoded factor was identified from three pairs of complementary fragment ions (b/y) which were also used to obtain a more accurate calculation of the mass of the protein than that possible from MS analysis. A software application tool was developed to rapidly search amino acid sequences from whole genome sequencing-derived data to match the observed protein mass and fragment ions on the assumption that the fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of aspartic acid and glutamic acid residues, i.e. aspartic acid effect. Manual inspection and top-down proteomic analysis confirmed the correctness of the identification of this protein using this new application tool. Although the function of the plasmid-borne factor is not known, its antibiotic-induced expression may suggest a role in antimicrobial stress. This approach was also used to identify three highly conserved bacterial cold-shock proteins: CspC, CspE, CsbD from one of the STEC strains. In addition to polypeptide backbone cleavage, metastable CsbD showed multiple small dissociative losses perhaps suggesting ammonia loss from the four arginine residues in its sequence. This phenomenon had been previously observed for metastable ubiquitin which also has four arginine residues.