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.
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.
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710, USA. clifton.fagerquist@ars.usda.gov
Clifton K
CK
Fagerquist
Brandon R
BR
Garbus
William G
WG
Miller
Katherine E
KE
Williams
Emma
E
Yee
Anna H
AH
Bates
Síobhán
S
Boyle
Leslie A
LA
Harden
Michael B
MB
Cooley
Robert E
RE
Mandrell
D000465:Algorithms; D000595:Amino Acid Sequence; D015415:Biomarkers / Q000378:metabolism; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods*; D012703:Serotyping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D015415:Biomarkers; D029968:Escherichia coli Proteins