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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY AND GENOMICS OF FOODBORNE PATHOGENS

Location: Produce Safety and Microbiology Research

Title: Rapid Identification of Protein Biomarkers of E. coli O157:H7 by MALDI-TOF-TOF Mass Spectrometry and Top-Down Proteomics

Authors
item Fagerquist, Clifton
item Garbus, Brandon -
item Miller, William
item Williams, Katherine -
item Yee, Emma
item Bates, Anne
item Boyle, Siobhan
item Harden, Leslie
item Cooley, Michael
item Mandrell, Robert

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2010
Publication Date: April 1, 2010
Citation: Fagerquist, C.K., Garbus, B.R., Miller, W.G., Williams, K.E., Yee, E., Bates, A.H., Boyle, S., Harden, L.A., Cooley, M.B., Mandrell, R.E. 2010. Rapid Identification of Protein Biomarkers of E. coli O157:H7 by MALDI-TOF-TOF Mass Spectrometry and Top-Down Proteomics. Analytical Chemistry. 82:2717-2725.

Interpretive Summary: We have identified six protein biomarkers from two strains of E. coli O157:H7 and one non-pathogenic E. coli strain by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometry (TOF/TOF-MS/MS) and top-down proteomics. Mature, intact proteins were extracted from bacterial cell lysates, ionized by MALDI, mass-isolated, fragmented and analyzed by TOF/TOF-MS/MS. Protein 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 in-house developed software was used to rapidly compare mass-to-charge (m/z) of MS/MS fragment ions to m/z of in silico fragment ions from hundreds of bacterial protein sequences and score/rank identifications on the basis of the number in silico-MS/MS matches. A peak matching algorithm and a p-value algorithm were used to independently score/rank identifications. 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. The mature proteins of hdeA, hdeB, ybgS and yahO were found to be post-translationally modified with removal of a N-terminal signal peptide. Gene sequencing of hdeA, hdeBWe have identified six protein biomarkers from two strains of E. coli O157:H7 and one non-pathogenic E. coli strain by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometry (TOF/TOF-MS/MS) and top-down proteomics. Mature, intact proteins were extracted from bacterial cell lysates, ionized by MALDI, mass-isolated, fragmented and analyzed by TOF/TOF-MS/MS. Protein 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 in-house developed software was used to rapidly compare mass-to-charge (m/z) of MS/MS fragment ions to m/z of in silico fragment ions from hundreds of bacterial protein sequences and score/rank identifications on the basis of the number in silico-MS/MS matches. A peak matching algorithm and a p-value algorithm were used to independently score/rank identifications. 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. The mature proteins of hdeA, hdeB, ybgS and yahO were found to be post-translationally modified with removal of a N-terminal signal peptide. Gene sequencing of hdeA, hdeBWe have identified six protein biomarkers from two strains of E. coli O157:H7 and one non-pathogenic E. coli strain by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometry (TOF/TOF-MS/MS) and top-down proteomics. Mature, intact proteins were extracted from bacterial cell lysates, ionized by MALDI, mass-isolated, fragmented and analyzed by TOF/TOF-MS/MS. Protein 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 in-house developed software was used to rapidly compare mass-to-charge (m/z) of MS/MS fragment ions to m/z of in silico fragment ions from hundreds of bacterial protein sequences and score/rank identifications on the basis of the number in silico-MS/MS matches. A peak matching algorithm and a p-value algorithm were used to independently score/rank identifications. 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 put

Technical Abstract: We have identified six protein biomarkers from two strains of E. coli O157:H7 and one non-pathogenic E. coli strain by matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometry (TOF/TOF-MS/MS) and top-down proteomics. Mature, intact proteins were extracted from bacterial cell lysates, ionized by MALDI, mass-isolated, fragmented and analyzed by TOF/TOF-MS/MS. Protein 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 in-house developed software was used to rapidly compare mass-to-charge (m/z) of MS/MS fragment ions to m/z of in silico fragment ions from hundreds of bacterial protein sequences and score/rank identifications on the basis of the number in silico-MS/MS matches. A peak matching algorithm and a p-value algorithm were used to independently score/rank identifications. 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. The mature proteins of hdeA, hdeB, ybgS and yahO were found to be post-translationally modified with removal of a N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, yjbJ for eleven strains of E. coli O157:H7 and seven strains of the near-neighbor E. coli serotype O55:H7 revealed a high degree sequence homology between these two serotypes for these genes/proteins. Although it was not possible to distinguish O157:H7 from O55:H7 using these six protein biomarkers, it was possible to distinguish E. coli O157:H7 from non-pathogenic E. coli by top-down proteomics of the yahO and ybgS proteins. In the case of the yahO protein, a single amino acid substitution in the yahO sequence (aspartic acid for asparagine which resulted in a protein molecular weight difference of only 1 Da) was sufficient to distinguish between E. coli O157:H7 from non-pathogenic E. coli by MS/MS, whereas this would be difficult to distinguish by MS. Finally, a protein biomarker at m/z ' 9060, which is observed in the MALDI-TOF-MS spectra of non-O157:H7 E. coli strains but which is conspicuously absent from spectra of E. coli O157:H7 strains and which had been previously identified by gene sequencing and bottom-up proteomics, was confirmed by top-down analysis to be the acid stress chaperone-like protein: hdeB. cspC, ybgS, yahO, yjbJ for eleven strains of E. coli O157:H7 and seven strains of the near-neighbor E. coli serotype O55:H7 revealed a high degree sequence homology between these two serotypes for these genes/proteins. Although it was not possible to distinguish O157:H7 from O55:H7 using these six protein biomarkers, it was possible to distinguish E. coli O157:H7 from non-pathogenic E. coli by top-down proteomics of the yahO and ybgS proteins. In the case of the yahO protein, a single amino acid substitution in the yahO sequence (aspartic acid for asparagine which resulted in a protein molecular weight difference of only 1 Da) was sufficient to distinguish between E. coli O157:H7 from non-pathogenic E. coli by MS/MS, whereas this would be difficult to distinguish by MS. Finally, a protein biomarker at m/z ' 9060, which is observed in the MALDI-TOF-MS spectra of non-O157:H7 E. coli strains but which is conspicuously absent from spectra of E. coli O157:H7 strains and which had been previously identified by gene sequencing and bottom-up proteomics, was confirmed by top-down analysis to be the acid stress chaperone-like protein: hdeB.

Last Modified: 4/19/2014
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