Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of Campylobacter species and strains by MALDI-TOF-MS protein biornarker analysis
Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of Campylobacter species and strains by MALDI-TOF-MS protein biornarker analysis
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DOI：
10.1021/ac040193z
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发表时间：
2005-08-01
影响因子：
7.4
通讯作者：
Mandrell, RE
中科院分区：
文献类型：
--
作者：
Fagerquist, CK;Miller, WG;Mandrell, RE
我们在基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF-MS)中发现了一个与10 kDa蛋白质相似的生物标记物，这些生物标记物来自空肠、大肠杆菌、幼虫、上唾液杆菌和螺旋藻。基因组和蛋白质组测序明确地将该生物标记物确定为DNA结合蛋白HU。通过对空肠弯曲菌(2株)、大肠埃希氏菌(4株)、华支睾吸虫(4株)和华支睾吸虫(2株)的HUP基因序列测定，确定了HU的氨基酸序列。提取/消化的Hu蛋白的胰蛋白酶多肽通过纳流高效液相色谱-串联质谱仪进行了氨基酸序列的确认。通过比较HUP基因预测的相对分子质量和高分辨质谱仪测得的HU的相对分子质量，也证实了蛋白质的鉴定。我们发现Hu蛋白作为生物标记物特别有用，因为它被MALDI强烈电离，其分子量在物种之间和物种内的菌株之间有所不同。HU分子量的种内和种间差异是由于HU蛋白氨基酸序列的变化，而不是由于共翻译或翻译后修饰。MALDI对HU的强烈电离效率可能部分归因于蛋白质羧基末端聚集的四个赖氨酸残基。我们还报道了一株Helveticus菌株的HU蛋白生物标志物的鉴定，该菌株的HUP基因没有测序，但其HU氨基酸序列在华支睾吸虫菌株中部分保守。我们还初步将一株致密螺旋藻的10.5 kDa蛋白生物标志物命名为安虎蛋白。
We have identified a prominent similar to 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 similar to 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.