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

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.

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

“…However, some recent studies have demonstrated that the noncovalent dissociation in the matrix‐assisted laser desorption/ionization (MALDI) process can be overcome by the use of appropriate matrix and chemical cross‐linking (Chen et al, 2016). The potential of MALDI combined with TDMS for proteomics studies has also been explored (Fagerquist & Dodd, 2021; Fagerquist & Sultan, 2011; Oros et al, 2020; Sturiale et al, 2011). Moreover, a laser ablation electrospray ionization (LAESI) was demonstrated for ionizing protein complexes in their native state (Kooijman et al, 2021).…”

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research

“…MALDI-ToF/ToF was used as an analytical tool for the determination of bacteria in urine samples collected from patients with urinary tract infections [ 108 ]. Furthermore, MALDI-ToF/ToF was successfully used for identification of full and truncated proteins produced by pathogenic Escherichia coli strains [ 140 ]. MALDI-ToF/ToF MS could aid in the generation of accurate molecular formulas and structural information to rapidly discriminate bacterial function [ 141 ] as well as for identification of new viral evasion strategies and fundamental factors governing host-microbial interactions as has been demonstrated in HPV infection that alters vaginal microbiome via downregulation of host mucosal innate peptides used by Lactobacilli as amino acid sources [ 142 ].…”

Native ambient mass spectrometry of intact protein assemblies directly fromEscherichia colicolonies

“…Among those proteins, HdeA (monomer), HdeB (monomer), CspE and Hpr have previously been identified by MALDI top-down MS of cell lysates of various strains of E. coli . 25 HdeA, HdeB, YibT and YgiW were identified in a bottom-up LC-MS proteomics study of outer membrane vesicles obtained from a range of E. coli strains. 26 Acyl carrier protein has been identified in a bottom-up MALDI study.…”