Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9.,Journal of Proteome Research

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Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-03-05 , DOI: 10.1021/acs.jproteome.0c00072
Katarzyna H Kucharzyk ₁ , Jayda E Meisel ₁ , Fadime Kara-Murdoch _{2,

3} , Robert W Murdoch _{4,

5} , Steven A Higgins ₂ , Simon Vainberg ₆ , Craig M Bartling ₁ , Larry Mullins ₁ , Paul B Hatzinger ₆ , Frank E Löffler _{2,

3,

4,

5,

7}

Affiliation

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with ¹³C-/¹⁵N-labeled peptides determined 1.8 × 10³ TceA and 1.2 × 10² VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

中文翻译：

代谢组学的蛋白质组学定量的含Dehalococcoides mccartyi的财团SDC-9中的还原性脱卤素酶。

在被氯化乙烯污染的地下水位，经常添加可发酵的底物，以促进土著或增强微生物的还原性脱卤作用。当代的生物修复性能监测依赖于关键的可吸入有机卤化物的细菌的核酸生物标记物，例如Dehalococcoides mccartyi（Dhc）。商业的，含Dhc的财团SDC-9的元基因组测序确定了12个还原性脱卤素酶（RDase）基因，包括pceA（两个副本），vcrA和tceA，并允许使用液相色谱和串联质谱（LC-MS / MS）进行RDase肽的特异性检测和定量。应用于与四氯乙烯（PCE）和乳酸盐一起生长的SDC-9财团的弹枪（即非靶向）蛋白质组学鉴定了143种RDase肽和36种不同的肽，它们覆盖了PceA，TceA和PceA的蛋白质编码序列的99％以上VcrA RDase。使用¹³ C- / ¹⁵ N标记的肽的多反应监测（MRM）分析法对RDase肽进行定量，确定每个Dhc细胞1.8×10 ³ TceA和1.2×10 ² VcrA RDase分子。MRM质谱方法可实现灵敏的检测和相关物质的准确定量Dhc RDases并在生物修复监测体系中具有潜在的实用性。

更新日期：2020-04-24

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