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
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- 作者：Clifton K. Fagerquist ; William G. Miller ; Leslie A. Harden ; Anna H. Bates ; William H. Vensel ; Guilin Wang ; and Robert E. Mandrell
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：August 1, 2005
- 年：2005
- 卷：77
- 期：15
- 页码：4897 - 4907
- 全文大小：680K
- 年卷期：v.77,no.15(August 1, 2005)
- ISSN：1520-6882
文摘
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 genomicand proteomic sequencing as a DNA-binding protein HU.We report the amino acid sequence of HU as determinedby 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 peptidesof the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecularweight (MW) predicted from the hup gene and the MWof HU as measured by high-resolution mass spectrometry.We found the HU protein to be particularly useful as abiomarker in that it strongly ionizes by MALDI and its MWvaries between species and among strains within aspecies. Intra- and interspecies variation of the HU MWis due to changes in the amino acid sequence of the HUprotein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI islikely due, in part, to four lysine residues clustered at thecarboxyl end of the protein. We also report identificationof the HU protein biomarker for a C. helveticus strain,whose hup gene was not sequenced, but whose HU aminoacid sequence was partially conserved in C. upsaliensisstrains. We have also tentatively assigned a ~10.5-kDaprotein biomarker of a C. concisus strain as an HUprotein.