Rapid Identification of Protein Biomarkers of Escherichia coli 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.

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…A number of prominent peaks are observed, which correspond to high copy, cytosolic, or periplasmic proteins. For example, the peaks at m/z 7712, 9742, and 10477 have been identified in other E. coli strains as the YahO protein, the acid stress protein HdeA, and the homeobox protein YbgS, respectively [26]. Figure 2b shows an expanded mass range from Figure 2a.…”

“…Mass spectrometry (MS) and tandem mass spectrometry with post-source decay (MS/MS-PSD) data was collected using a 4800 MALDI-TOF-TOF mass spectrometer (AB SCIEX, Framingham, MA, USA) as described previously [24][25][26]. Briefly, cells were harvested from the agar plate after overnight culturing (17 h) using a sterile plastic 1.0 μL microbiological loop, which when filled corresponded to approximately 1×10 9 cells [24].…”

“…Both bottom-up and top-down have certain advantages and disadvantages and the approach taken is often dependent on the instrumentation and personnel available and the objectives of the research. We have previously demonstrated a rapid technique for identification of protein biomarkers from unfractionated bacterial cell lysates using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight mass spectrometry (MALDI-TOF-TOF-MS) and top-down proteomic analysis [24][25][26]. We also demonstrated this approach in the identification of the B-subunit and the A2 fragment of the Asubunit of Stx2 in E. coli O157:H7 strain EDL933 by antibiotic induction [27,28].…”

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

“…Samples were prepared for MALDI analysis as described previously [34][35][36]. Briefly, a 1 μL sterile loop was used to harvest cells from each plate after overnight culturing and transferred to an O-ring lined, 2 mL screw-cap tube containing 300 μL of extraction solution (either 300 μL water or 300 μL of 67 % water, 33 % acetonitrile and 0.2 % trifluoroacetic acid [TFA]) and~40 mg of 0.1 mm zirconia/ silica beads.…”

“…Although initially developed as a high throughput platform for bottom-up proteomics, MALDI-TOF-TOF instruments are increasingly utilized for top-down proteomic identification of non-digested proteins using either in-source decay (ISD) [28][29][30] or post-source decay (PSD) [31][32][33][34][35][36]. As ISD and PSD occur in different regions of the mass spectrometer there are significant differences in ion fragmentation and polypeptide backbone cleavage.…”