Proteogenomic Definition of Biomarkers for the Large <i>Roseobacter</i> Clade and Application for a Quick Screening of New Environmental Isolates

Whole-cell, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become a routine and reliable method for microbial characterization due to its simplicity, low cost, and high reproducibility. The identification of microbial isolates relies on the spectral resemblance of low-molecular-weight proteins to already-existing isolates within the databases. This is a gold standard for clinicians who have a finite number of well-defined pathogenic strains but represents a problem for environmental microbiologists with an overwhelming number of organisms to be defined. Here we set a milestone for implementing whole-cell MALDI-TOF mass spectrometry to identify isolates from the biosphere. To make this technique accessible for environmental studies, we propose to (i) define biomarkers that will always show up with an intense m/z signal in the MALDI-TOF spectra and (ii) create a database with all the possible m/z values that these biomarkers can generate to screen new isolates. We tested our method with the relevant marine Roseobacter lineage. The use of shotgun nanoLC-MS/MS proteomics on the small proteome fraction of nine Roseobacter strains and the proteogenomic toolbox helped us to identify potential biomarkers in terms of protein abundance and low variability among strains. We show that the DNA binding protein, HU, and the ribosomal proteins, L29 and L30, are the most robust biomarkers within the Roseobacter clade. The molecular weights of these three biomarkers, as for other conserved homologous proteins, vary due to sequence variation above the genus level. Therefore, we calculated the m/z values expected for each one of the known Roseobacter genera and tested our strategy during an extensive screening of natural marine isolates obtained from coastal waters of the Western Mediterranean Sea. The use of this technique versus standard sequencing methods is discussed.全细胞基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱因操作简便、成本低廉且重现性优异，已成为微生物鉴定的常规可靠手段。微生物分离株的鉴定依赖于低分子量蛋白质谱与数据库中已有分离株的谱图相似性。对于仅需处理有限株数明确致病菌株的临床医师而言，该方法堪称金标准；但对于需鉴定海量微生物的环境微生物学家来说，却构成了一大难题。本研究为利用全细胞MALDI-TOF质谱鉴定生物圈来源的分离株树立了重要里程碑。为使该技术可应用于环境研究，我们提出两项方案：（i）定义在MALDI-TOF谱图中始终会产生强质荷比（m/z）信号的生物标志物；（ii）构建包含这些生物标志物所能产生的全部可能m/z值的数据库，用于新分离株的筛选。我们以具有代表性的海洋玫瑰杆菌谱系对该方法进行了验证。通过对9株玫瑰杆菌的小蛋白质组组分开展鸟枪法纳升液相色谱-串联质谱（shotgun nanoLC-MS/MS）蛋白质组学分析，并借助蛋白基因组学工具包，我们鉴定出了在菌株间丰度较高且变异性较低的潜在生物标志物。研究表明，DNA结合蛋白HU以及核糖体蛋白L29、L30是玫瑰杆菌分支中稳定性最优的生物标志物。与其他保守同源蛋白类似，这三种生物标志物的分子量会因属水平以上的序列变异而存在差异。因此，我们计算了已知玫瑰杆菌各属的预期m/z值，并在对西地中海沿岸海域获取的大量天然海洋分离株开展的大规模筛选中验证了该策略。本文还讨论了该技术与标准测序方法的应用对比。