Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1
Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1
复制标题
稳定同位素肽质谱法破译 Dehalococcoides 菌株 CBDB1 的氨基酸代谢
DOI：
10.1128/jb.00049-12
复制
发表时间：
2012
影响因子：
3.2
通讯作者：
L. Adrian
中科院分区：
文献类型：
--
作者：
Marco-Urrea;J. Seifert;M. von Bergen;L. Adrian
Dehalococcoides物种是受污染场地卤化溶剂厌氧转化的关键参与者。在这里，我们分析同位素分布在氨基酸池的肽Dehalococcoides菌株CBDB 1与13 C-标记的醋酸盐或碳酸氢盐作为碳源孵育后。对所得数据进行基因组注释以鉴定氨基酸生物合成途径。除了使用气相色谱-质谱（GC-MS）分析蛋白质水解后衍生的氨基酸，我们引入了第二种，温和得多的方法，其中我们直接分析胰蛋白酶消化后的肽质量和肽片段的纳米液相色谱-电喷雾电离-串联质谱（nano-LC-ESI-MS/MS）。用这种方法，我们确定了17个蛋白质氨基酸，包括脯氨酸，半胱氨酸，赖氨酸和精氨酸，逃脱了以前的分析Dehalococcoides同位素掺入模式。我们的研究结果证实了赖氨酸的生物合成通过α-氨基己二酸途径，排除了赖氨酸的形成从天冬氨酸。同样，精氨酸的同位素模式提供了其从谷氨酸合成的生化证据。直接肽MS/MS分析的标记模式的谷氨酰胺和天冬酰胺，这是转化为谷氨酸和天冬氨酸蛋白质水解过程中，给出了生化证据，其前体和确认谷氨酸生物合成通过aRe特异性柠檬酸合酶。通过将未标记的游离氨基酸添加到标记的细胞中，我们表明在菌株CBDB 1中，17种测试的氨基酸中没有一种被掺入细胞团中，表明它们都是从头合成的。我们的方法是广泛适用的，并提供了一种手段，通过研究特定的蛋白质，甚至在混合财团分析氨基酸代谢。
Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with13C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via aRe-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesizedde novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.
登录
查看更多内容
影响因子：
11.4
作者：
Marco-Urrea, Ernest;Nijenhuis, Ivonne;Adrian, Lorenz
通讯作者：
Adrian, Lorenz
影响因子：
7
作者：
Nishida, H;Nishiyama, M;Yamane, H
通讯作者：
Yamane, H
嗜中性嗜热变形杆菌中乙酸同化的 13C-NMR 研究。
DOI：
--
发表时间：
1989
期刊：
European Journal of Biochemistry
影响因子：
--
作者：
S. Schäfer;T. Paalme;R. Vilu;G. Fuchs
通讯作者：
G. Fuchs
影响因子：
3.2
作者：
Risso, Carla;Van Dien, Stephen J.;Coppi, Maddalena V.
通讯作者：
Coppi, Maddalena V.
螺旋体钩端螺旋体中的氨基酸生物合成：异亮氨酸生物合成新途径的证据
影响因子：
3.2
作者：
N. Charon;R. C. Johnson;D. Peterson
通讯作者：
D. Peterson