Location: Produce Safety and Microbiology ResearchTitle: 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
Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2018
Publication Date: 12/14/2018
Citation: Fagerquist, C.K., Lee, B.G., Zaragoza, W.J., Yambao, J.C., Quiñones, B. 2018. 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. International Journal of Mass Spectrometry. 438:1-12. https://doi.org/10.1016/j.ijms.2018.12.006.
Interpretive Summary: Rapid methods are needed to identify and characterize pathogenic bacteria. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is increasingly utilized for taxonomic identification of bacteria including foodborne pathogens. This approach has been further extended to the identification of bacterial proteins using a dual TOF instrument (TOF-TOF) that allows isolation, fragmentation and identification of specific proteins and toxins. The MALDI-TOF-TOF top-down proteomic approach has a number of advantages. First, the sample preparation is simple. Second, data acquisition is extremely rapid requiring only seconds to minutes. Third, data analysis is similarly rapid if bacterial protein sequences are publically available or have been previously assembled in-house. We have identified a plasmid-encoded factor (hypothetical protein) in three Shiga toxin Escherichia coli (STEC) strains that is highly expressed when the STEC strain is exposed to a DNA-damaging antibiotic during culturing. Tandem mass spectrometry (MS/MS) of this unknown protein produced fragment ions consisting of a series of complementary b/y fragment ions that allowed a second, more accurate measurement of the mass of the protein. Using the measured/calculated protein mass and assuming that MS/MS fragment ions are the result of polypeptide backbone cleavage on the C-terminal side of an aspartic acid or glutamic acid residues, a software application tool was developed that searches the open reading frames of the bacterial genome to find the corresponding protein. This search resulted in the identification of a hypothetical protein whose gene resides in a plasmid. The correctness of this sequence was confirmed by manual inspection and top-down proteomic analysis. Although the function of this protein is not known, it expression as a result of antibiotic exposure may suggest a role in antimicrobial stress.
Technical Abstract: 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.