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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #174977
Title: GENOMIC AND PROTEOMIC IDENTIFICATION OF A DNA-BINDING PROTEIN USED IN THE "FINGERPRINTING" OF CAMPYLOBACTER SPECIES AND STRAINS BY MALDI-TOF-MS PROTEIN BIOMARKER ANALYSIS

Author
item Fagerquist, Clifton - Keith
item Miller, William - Bill
item Harden, Leslie - Les
item Bates, Anne
item Vensel, William
item Wang, Guilin
item Mandrell, Robert

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2005
Publication Date: 8/1/2005
Citation: Fagerquist, C.K., Miller, W.G., Harden, L.A., Bates, A.H., Vensel, W.H., Wang, G., Mandrell, R.E. 2005. Genomic and proteomic identification of a dna-binding protein used in the "fingerprinting" of campylobacter species and strains by maldi-tof-ms protein biomarker analysis. Analytical Chemistry. web release 6/25/05, 77:4897-4907

Interpretive Summary: The rapid detection and analysis of human pathogens is an increasingly important area of analytical science that has been spurred by recent concerns about biosecurity, however pathogen detection and characterization has always been important for food safety. Sensitivity, specificity, speed, simplicity and ruggedness are all critical objectives in the improvement of existing methods of detection and the development of new techniques. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a powerful tool for the rapid detection and characterization of microorganisms on the basis of their unique protein biomarkers. An important human pathogen is Campylobacter which has been estimated to cause ~ 2.4 million incidents of food-borne illness every year in the US alone, in addition to being a significant health problem worldwide. We have identified a prominent ~10 kDa protein biomarker observed in the MALDI-TOF-MS spectra of cell lysates of five species of Campylobacter: C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein: HUP. We found the HUP protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its molecular weight (MW) varies between species (and among strains within a species) due to changes in its primary amino acid sequence. We report the primary HUP sequence (determined from genetic sequencing) for four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari (2). Confirmation of the primary sequence was obtained by nano-flow high performance liquid chromatography-tandem mass spectrometry of the extracted/digested HUP protein. Protein confirmation was also obtained by comparison of the predicted and measured HUP MW.

Technical Abstract: We have identified a prominent ~10 kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein: HUP. We found the HUP protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its molecular weight (MW) varies between species and among strains within a species. We report the primary HUP amino acid sequence as determined by sequencing of the hup gene for four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari (2). Confirmation of the primary sequence was obtained by nano-flow high performance liquid chromatography-tandem mass spectrometry of the extracted, trypsin-digested protein. Protein confirmation was also obtained by comparison of the genetically predicted HUP MW and the measured protein MW obtained by high resolution mass spectrometry. Intra- and inter-species variation of the HUP MW is due to changes in the genetically determined primary amino acid sequence of the protein and not due to co- or post-translational modifications. The strong ionization efficiency of HUP by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HUP biomarker for a C. helveticus strain, whose HUP gene was not sequenced, but whose HUP primary sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a ~ 10.5 kDa protein biomarker from a C. concisus strain as the HUP protein.