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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 E. coli O157:H7 by "Top-Down" Proteomics Using MALDI-TOF/TOF Mass Spectrometry

Authors
item Fagerquist, Clifton
item Garbus, Brandon - INTUITIVE SURGICAL
item Williams, Katherine - UCSF SCH. OF MEDICINE
item Bates, Anne
item Boyle, Siobhan
item Harden, Leslie
item Miller, William
item Mandrell, Robert

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 10, 2009
Publication Date: N/A

Technical Abstract: This abstract was presented as an oral presentation on June 2nd 2009 at the 57th American Society of Mass Spectrometry Conference (May 31-June 4, 2009, Philadelphia, PA). Rapid identification of bacterial microorganisms is of importance for food safety and security. Mass spectrometry is at the forefront of the identification and characterization of bacteria and specifically foodborne pathogens. Matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF-TOF) mass spectrometers have been shown to fragment modest-sized proteins generating sequence-specific fragment ions that can be used for identification of the protein (and its source microorganism) without digestion. We have identified several unique protein biomarker sequences from two strains of E. coli O157:H7 (a strain isolated from Salinas River, CA and the EDL-933 reference strain) and one non-pathogenic, non-O157:H7 E. coli strain using "top-down" proteomics MALDI-TOF-TOF-MS/MS. Mature, intact protein biomarkers were ionized, mass-selected, fragmented and the fragment ions detected. Proteins were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from thousand of bacterial protein sequences using in-house developed software. The putative uncharacterized protein yahO and the putative homeobox protein were found to have amino acid sequences unique to O157:H7 strains. The amino acid sequence of the yahO protein of EDL-933 is different from that of the non-pathogenic E. coli strain by a single amino acid substitution, aspartic acid ' asparagine (D ' N), resulting in a protein MW difference of only 1 Da which is difficult to distinguish by MALDI-TOF-MS. However, "top-down" MS/MS clearly distinguishes these two protein sequences and their source microorganisms. We also confirmed the identity by "top-down" analysis of a protein associated with acid-resistance (hdeB) which appears in the MALDI-TOF-MS spectra of non-O157:H7 E. coli strains but which is absent from spectra of O157:H7 strains due to a mis-sense mutation in the start codon of the hdeB gene resulting in this protein biomarker not being expressed. Finally, we report the covalent attachment (via a thio-ester linkage) of the MALDI matrix 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) to cysteine-containing protein biomarkers resulting in additional peaks in the MALDI-TOF-MS spectra when using this matrix. Such additional peaks were not observed when using '-cyano-4-hydroxycinnamic acid (HCCA). This differential reactivity of MALDI matrices may be exploited for identification of unknown cysteine-containing protein biomarkers.

Last Modified: 7/14/2014
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