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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: Identification of Foodborne Bacteria by High Energy Collision-Induced Dissociation of Their Protein Biomarkers by MALDI Tandem-Time-of-Flight Mass Spectrometry

Authors
item Fagerquist, Clifton
item Williams, Katherine - APPLIED BIOSYSTEMS
item Bates, Anne

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Proceedings
Publication Acceptance Date: February 2, 2007
Publication Date: June 7, 2007
Citation: Fagerquist, C.K., Williams, K.E., Bates, A. H. Identification of Foodborne Bacteria by High Energy Collision-Induced Dissociation of their Protein Biomarkers by MALDI Tandem-Time-of-Flight Mass Spectrometry. In: Proceedings of the 55th American Society of Mass Spectrometry Conference, June 3-7, 2007, Indianapolis, IN. [DVD MPT-327 from ASMS.]

Interpretive Summary: Development of methods for rapid identification of foodborne bacteria is an important area of analytical science and food safety. MALDI-TOF-MS has been utilized to rapidly identify pathogens including foodborne bacteria. Identification typically involves detection of high copy cytosolic proteins in the mass range 3-20 kDa. Data analysis uses either pattern recognition or bioinformatic algorithms. With bioinformatic algorithms, protein biomarkers are tentatively identified (if at all) on the basis of mass-to-charge (m/z) although this approach is problematical because of multiple proteins with the same molecular weight or the presence of post-translational modifications. With the advent of tandem MALDI instruments (TOF-TOF), it is possible to fragment modest-sized proteins to obtain sequence-specific information potentially allowing definitive identification of both the protein as well as the pathogen. We have analyzed the protein biomarkers from cell lysates of four genomically sequenced bacterial strains which are representative of four species of Campylobacter: C. jejuni, C. coli, C. upsaliensis and C. lari. Protein biomarkers (i.e. 50S ribosomal L7/L12, thioredoxin, protein of unknown function DUF465, cytochrome c, 10 kD chaperonin) from the un-fractionated lysate were ionized by matrix-assisted laser desorption/ionization (MALDI), mass-selected with a time-of-flight (TOF) mass gate, fragmented by high energy collision-induced dissociation (HE-CID, > 1.5 keV) and the fragment ions analyzed by the second TOF mass analyzer. Various MALDI matrices were tested for their ability to ionize as well as facilitate the fragmentation process. The significant findings are as follows. 1. The fragment ions detected are primarily b and y ions that are the result of cleavage of the polypeptide chain adjacent to aspartic and glutamic acid residues. Proton transfer from the side-chains of these acidic residues to the amide bond of the peptide backbone is responsible for fragmentation of the polypeptide. The relatively short side-chain length of aspartic acid may facilitate peptide backbone cleavage adjacent to this residue. 2. Variations in the protein amino acid sequence across species/strains result in "shifts" in the m/z of observed fragment ions. 3. Amino acid substitutions that involve an aspartic or glutamic acid residue can also significantly effect the fragmentation pathways of a protein determining both the type of ion formed as well as the site of polypeptide cleavage. 4. Facile loss of the covalently-bound heme group was observed for HE-CID of cytochrome-c. 5. MALDI-tandem-TOF analysis of bacterial proteins provides sequence-specific information which may be used by proteomic/bioinformatic algorithms to identify protein biomarkers and the pathogen which produced them. This experiment represents a rapid and simple "top-down" proteomic approach to bacterial protein biomarker identification.

Technical Abstract: Development of methods for rapid identification of foodborne bacteria is an important area of analytical science and food safety. MALDI-TOF-MS has been utilized to rapidly identify pathogens including foodborne bacteria. Identification typically involves detection of high copy cytosolic proteins in the mass range 3-20 kDa. Data analysis uses either pattern recognition or bioinformatic algorithms. With bioinformatic algorithms, protein biomarkers are tentatively identified (if at all) on the basis of mass-to-charge (m/z) although this approach is problematical because of multiple proteins with the same molecular weight or the presence of post-translational modifications. With the advent of tandem MALDI instruments (TOF-TOF), it is possible to fragment modest-sized proteins to obtain sequence-specific information potentially allowing definitive identification of both the protein as well as the pathogen. We have analyzed the protein biomarkers from cell lysates of four genomically sequenced bacterial strains which are representative of four species of Campylobacter: C. jejuni, C. coli, C. upsaliensis and C. lari. Protein biomarkers (i.e. 50S ribosomal L7/L12, thioredoxin, protein of unknown function DUF465, cytochrome c, 10 kD chaperonin) from the un-fractionated lysate were ionized by matrix-assisted laser desorption/ionization (MALDI), mass-selected with a time-of-flight (TOF) mass gate, fragmented by high energy collision-induced dissociation (HE-CID, > 1.5 keV) and the fragment ions analyzed by the second TOF mass analyzer. Various MALDI matrices were tested for their ability to ionize as well as facilitate the fragmentation process. The significant findings are as follows. 1. The fragment ions detected are primarily b and y ions that are the result of cleavage of the polypeptide chain adjacent to aspartic and glutamic acid residues. Proton transfer from the side-chains of these acidic residues to the amide bond of the peptide backbone is responsible for fragmentation of the polypeptide. The relatively short side-chain length of aspartic acid may facilitate peptide backbone cleavage adjacent to this residue. 2. Variations in the protein amino acid sequence across species/strains result in "shifts" in the m/z of observed fragment ions. 3. Amino acid substitutions that involve an aspartic or glutamic acid residue can also significantly effect the fragmentation pathways of a protein determining both the type of ion formed as well as the site of polypeptide cleavage. 4. Facile loss of the covalently-bound heme group was observed for HE-CID of cytochrome-c. 5. MALDI-tandem-TOF analysis of bacterial proteins provides sequence-specific information which may be used by proteomic/bioinformatic algorithms to identify protein biomarkers and the pathogen which produced them. This experiment represents a rapid and simple "top-down" proteomic approach to bacterial protein biomarker identification.

Last Modified: 8/22/2014
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