%0 Journal Article %A Fagerquist, Clifton K. %A Garbus, Brandon R. %A Miller, William G. %A Williams, Katherine E. %A Yee, Emma %A Bates, Anna H. %A Boyle, Síobhán %A Harden, Leslie A. %A Cooley, Michael B. %A Mandrell, Robert E. %D 2010 %T 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 %U https://acs.figshare.com/articles/journal_contribution/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/2780704 %R 10.1021/ac902455d.s001 %2 https://acs.figshare.com/ndownloader/files/4475116 %K coli %K acid residue substitution %K protein biomarker ion %K silico fragment ions %K degree sequence homology %K MS spectra %K strain %K protein sequences %K gene sequencing %K MALDI %X 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 ∼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. %I ACS Publications