Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry

Authors: Fagerquist, Clifton - Keith; Dodd, Claire

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/12/2021
Publication Date: 11/29/2021
Citation: Fagerquist, C.K., Dodd, C.E. 2021. Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry. PLoS ONE. 16(11). Article e0260650. https://doi.org/10.1371/journal.pone.0260650.
DOI: https://doi.org/10.1371/journal.pone.0260650

Interpretive Summary: Rapid identification of protein biomarkers of pathogenic bacteria helps in assessing their potential to survive and cause disease. MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis is a rapid method to identify intact proteins produced by bacteria. A number of proteins produced by three pathogenic E. coli strains have been identified using antibiotic induction, MALDI-TOF-TOF-MS/MS and top-down analysis. Of particular interest was the identification of bactericidal immunity proteins whose genes are located on a plasmid carried by the host in two of the three E. coli strains. These plasmid-borne proteins provide a competitive survival advantage to their host over other bacteria. A protein important as a global regulator of carbon metabolism was also identified. Many of the proteins identified have a modification involving sequence truncation in the mature protein. Our in-house top-down software successfully identified these truncated proteins, and the truncations were in nearly all cases consistent with those predicted by bioinformatic software.

Technical Abstract: Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.