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.
We have identified a prominent approximately 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 HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU 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 HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.
Fagerquist
Clifton K
CK
Western Regional Research Service, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, USA. cfagerquist@pw.usdsa.gov
Miller
William G
WG
Harden
Leslie A
LA
Bates
Anna H
AH
Vensel
William H
WH
Wang
Guilin
G
Mandrell
Robert E
RE
D000595:Amino Acid Sequence; D015415:Biomarkers / Q000032:analysis; D002167:Campylobacter / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D023281:Genomics / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D004268:DNA-Binding Proteins