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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: Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. concisus for bacterial identification

Authors
item FAGERQUIST, CLIFTON
item YEE, EMMA
item MILLER, WILLIAM

Submitted to: Analyst
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 22, 2007
Publication Date: July 20, 2007
Citation: Fagerquist, C.K., Yee, E., Miller, W.G. 2007. Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. consisus for bacterial identification. The Analyst. 132(10):1010-1023.

Interpretive Summary: Proteins biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of three strains of Campylobacter coli, two strains of C. lari and one strain of C. concisus have been identified by "bottom-up" proteomic techniques. The significant findings are as follows. First, the protein biomarkers identified were: DUF465 (protein of unknown function), phnA protein, 4-oxalocrotonate tautomerase (dmpI)-related protein, NifU-like protein, cytochrome c, DNA-binding protein HU, 10 kD chaperonin, thioredoxin, as well as several conserved hypothetical and ribosomal proteins. Second, variations in protein biomarker ion m/z observed in MALDI-TOF-MS spectra across species and strains are the result of variations in the amino acid sequence of the protein presumably due to non-synonymous mutations of the biomarker gene. Third, the most common post-translational modifications (PTM) were removal of N-terminal methionine and N-terminal signal peptides. However, in the case of NifU protein (an iron-sulfur cluster transport protein), post-translational cleavage occurred from the C-terminus. Fourth, only C. coli (strain RM2228) and C. lari (strain RM2100) have been sequenced genomically, and proteomic identification of the protein biomarkers of the other strains in this study relied upon partial sequence homology to genomically sequenced strains as well as other sequenced strains of Campylobacter. In some cases, determination of the full amino acid sequence of a protein biomarker from a genomically un-sequenced strain was accomplished by combining proteomic identifications of partially confirmed sequences from genomically sequenced strains of the same species and different species of Campylobacter. The correctness of this composite sequence was confirmed by MS and MS/MS. In some cases, it was necessary to perform de novo sequencing on regions of the biomarker sequence that were not homologous to any genomic sequence. As a result of this process, it was possible to determine the full amino acid sequence of an unknown protein biomarker from a genomically un-sequenced bacterial strain without the necessity of either DNA sequencing the biomarker gene or performing full de novo MS/MS sequencing of the protein. Among other uses, the full sequence can be used for "top-down" proteomics identification of protein biomarkers of foodborne pathogens.

Technical Abstract: Proteins biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of three strains of Campylobacter coli, two strains of C. lari and one strain of C. concisus have been identified by "bottom-up" proteomic techniques. The significant findings are as follows. First, the protein biomarkers identified were: DUF465 (protein of unknown function), phnA protein, 4-oxalocrotonate tautomerase (dmpI)-related protein, NifU-like protein, cytochrome c, DNA-binding protein HU, 10 kD chaperonin, thioredoxin, as well as several conserved hypothetical and ribosomal proteins. Second, variations in protein biomarker ion m/z observed in MALDI-TOF-MS spectra across species and strains are the result of variations in the amino acid sequence of the protein presumably due to non-synonymous mutations of the biomarker gene. Third, the most common post-translational modifications (PTM) were removal of N-terminal methionine and N-terminal signal peptides. However, in the case of NifU protein (an iron-sulfur cluster transport protein), post-translational cleavage occurred from the C-terminus. Fourth, only C. coli (strain RM2228) and C. lari (strain RM2100) have been sequenced genomically, and proteomic identification of the protein biomarkers of the other strains in this study relied upon partial sequence homology to genomically sequenced strains as well as other sequenced strains of Campylobacter. In some cases, determination of the full amino acid sequence of a protein biomarker from a genomically un-sequenced strain was accomplished by combining proteomic identifications of partially confirmed sequences from genomically sequenced strains of the same species and different species of Campylobacter. The correctness of this composite sequence was confirmed by MS and MS/MS. In some cases, it was necessary to perform de novo sequencing on regions of the biomarker sequence that were not homologous to any genomic sequence. As a result of this process, it was possible to determine the full amino acid sequence of an unknown protein biomarker from a genomically un-sequenced bacterial strain without the necessity of either DNA sequencing the biomarker gene or performing full de novo MS/MS sequencing of the protein. Among other uses, the full sequence can be used for "top-down" proteomics identification of protein biomarkers of foodborne pathogens.

Last Modified: 9/10/2014
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