Rapid Identification of Protein Biomarkers of <i>Escherichia coli</i> O157:H7 by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight−Time-of-Flight Mass Spectrometry and Top-Down Proteomics FagerquistClifton K. GarbusBrandon R. MillerWilliam G. WilliamsKatherine E. YeeEmma BatesAnna H. BoyleSíobhán HardenLeslie A. CooleyMichael B. MandrellRobert E. 2010 Six protein biomarkers from two strains of <i>Escherichia coli</i> O157:H7 and one non-O157:H7, nonpathogenic strain of <i>E. coli</i> 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 (<i>m</i>/<i>z</i>) of MS/MS fragment ions to the <i>m</i>/<i>z</i> of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a <i>p</i>-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 <i>hdeA</i>, <i>hdeB</i>, <i>cspC</i>, <i>ybgS</i>, <i>yahO</i>, and <i>yjbJ</i> for 11 strains of <i>E. coli</i> 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 <i>E. coli</i> O157:H7 from a nonpathogenic <i>E. coli</i> 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 <i>E. coli</i> O157:H7 from a non-O157:H7, nonpathogenic <i>E. coli</i> by MALDI-TOF−TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at <i>m</i>/<i>z</i> ∼9060 observed in the MS spectra of non-O157:H7 <i>E. coli</i> strains but absent from MS spectra of <i>E. coli</i> 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.