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Updated: Oct 11, 2025
Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis
Published on: May 23, 2021
Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using
Clifton K Fagerquist1, Claire E Dodd1
1Produce Safety & Microbiology, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California, United States of America.
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.
Insights
This study identified fourteen pathogenic Escherichia coli proteins using advanced mass spectrometry. The method accurately identified full and truncated bacterial proteins, including post-translational modifications and disulfide bonds.
Area of Science:
- Proteomics
- Microbiology
- Biochemistry
Background:
- Pathogenic Escherichia coli poses significant health risks.
- Accurate identification of bacterial proteins is crucial for understanding pathogenesis.
- Advanced proteomic techniques are needed to analyze complex bacterial proteomes.
Purpose of the Study:
- To identify proteins produced by pathogenic Escherichia coli strains.
- To evaluate the utility of MALDI-TOF-TOF tandem mass spectrometry for bacterial protein analysis.
- To characterize post-translational modifications and protein truncations in pathogenic E. coli.
Main Methods:
- Antibiotic induction of pathogenic Escherichia coli strains.
- Top-down proteomic analysis using MALDI-TOF-TOF tandem mass spectrometry (MS/MS).
- In-house software for data analysis, including protein sequence truncation and comparison with in silico sequences from whole genome sequencing.
Main Results:
- Identification of fourteen proteins from three pathogenic E. coli strains, including host and plasmid proteins.
- Characterization of post-translational modifications in nine proteins (N-terminal methionine removal or signal peptide cleavage).
- Accurate identification of full and truncated protein sequences by custom software, with high concordance to SignalP predictions.
Conclusions:
- MALDI-TOF-TOF tandem mass spectrometry is effective for identifying full and truncated bacterial proteins.
- The developed software accurately identifies protein truncations and post-translational modifications.
- This approach aids in the comprehensive analysis of pathogenic bacterial proteomes.
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