Rapid Identification of Protein Biomarkers of Escherichia coil O157:H7 by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight-Time-of-Flight Mass Spectrometry and Top-Down Proteomics
ABSTRACT Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.
SourceAvailable from: William J Zaragoza[Show abstract] [Hide abstract]
ABSTRACT: We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.Journal of the American Society for Mass Spectrometry 02/2015; DOI:10.1007/s13361-015-1076-3 · 3.19 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Rapid and efficient characterization and identification of pathogens at the strain level is of key importance for epidemiologic investigations, which still remains a challenge. In this work, solvothermically Fe3O4-COOH@MIL-101 composites were fabricated by in situ crystallization approach. The composites combine the excellent properties of both chromium (III) terephthalate (MIL-101) and carboxylic-functionalized magnetite (Fe3O4-COOH) particles and possess the efficient peptides/proteins enrichment properties and magnetic responsiveness. Fe3O4-COOH@MIL-101 composites as magnetic solid phase extraction materials were used to increase the discriminatory power of MALDI-TOF MS profiles. BSA tryptic peptides at a low concentration of 0.25fmolμL(-1) could be detected by MALDI-TOF MS. In addition, Fe3O4-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and discrimination of Escherichia coli at the strain level. This work provides the possibility for wide applications of magnetic MOFs to discriminate pathogens below the species level. Copyright © 2015 Elsevier B.V. All rights reserved.Analytica chimica acta 02/2015; 868. DOI:10.1016/j.aca.2015.02.018 · 4.52 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Matrix-assisted laser-desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass spectrometry based approaches for bacterial identification and classification. The relatively simple sample preparation requirements and the speed of analysis which can usually be completed within a few minutes have resulted in the adoption and assimilation of MALDI-TOF MS into the routine diagnostic workflow of Clinical microbiology laboratories worldwide. This study describes the facilitation of bacterial discrimination based on antibiotic resistance markers through the implementation of MALDI-TOF MS. The periplasmic compartment of whole bacterial cells contains several proteins which confer antibiotic resistance in the Enterobacteriaceae. In order to reduce the complexity of the sample to be analysed via MALDI-TOF MS, the periplasm was extracted and subjected to in solution tryptic digestion followed by nano-LC separation. This method, established that peptide sequence biomarkers from several classes of antibiotic resistance proteins could be predicted using protein/peptide database tools such as Mascot. Biomarkers for a group 1 CTX-M an extended spectrum β-lactamase, CMY-2 an Amp-C β-lactamase, VIM a metallo-β-lactamase, TEM a β-lactamase and KanR an aminoglycoside modifying enzyme were detected. This allowed for discrimination at a species level and at an almost identical strain level where the only difference between strains was the carriage of a modified antibiotic resistance carrying plasmid. This method also was able to detect some of these biomarkers in clinical strains where multiple resistance mechanisms were present. Copyright © 2015. Published by Elsevier B.V.Journal of Microbiological Methods 01/2015; 111. DOI:10.1016/j.mimet.2015.01.020 · 2.10 Impact Factor