Proteogenomic Definition of Biomarkers for the Large Roseobacter 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/电离飞行时间质谱（Whole-cell, matrix-assisted laser desorption/ionization time-of-flight, MALDI-TOF）质谱分析因其操作简便、成本低廉以及高度重现性，已成为微生物特征描述的常规且可靠的手段。微生物分离株的鉴定依赖于低分子量蛋白光谱与数据库中已存在分离株的相似性。对于具有有限且明确病原菌株的医生而言，此方法为金标准；然而，对于需要定义大量生物体的环境微生物学家而言，却构成了挑战。本研究旨在为生物圈中分离株的鉴定树立里程碑，通过实施全细胞MALDI-TOF质谱分析。为了使该技术适用于环境研究，我们提出以下策略：(i) 定义在MALDI-TOF光谱中始终呈现强烈m/z信号的生物标志物；(ii) 创建包含所有可能的m/z值的数据库，以筛选新的分离株。我们以相关的海洋Roseobacter类群为测试对象，通过针对九个Roseobacter菌株的小蛋白质组进行shotgun纳米液相色谱-质谱/质谱联用（nanoLC-MS/MS）蛋白质组学分析以及蛋白质组学工具箱，识别了以蛋白质丰度和菌株间低变异性为特征的潜在生物标志物。我们证实DNA结合蛋白HU和核糖体蛋白L29、L30是Roseobacter类群中最稳健的生物标志物。这些生物标志物的分子量，与其他保守同源蛋白类似，因属以上序列变异而有所不同。因此，我们计算了已知Roseobacter属中每个生物标志物的预期m/z值，并在对来自地中海西部沿海水域的天然海洋分离株的广泛筛选中测试了我们的策略。此外，我们还讨论了该技术与标准测序方法相比的优缺点。