Document Detail


Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1.
MedLine Citation:
PMID:  22661690     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 with (13)C-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 a Re-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 synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.
Authors:
Ernest Marco-Urrea; Jana Seifert; Martin von Bergen; Lorenz Adrian
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-06-01
Journal Detail:
Title:  Journal of bacteriology     Volume:  194     ISSN:  1098-5530     ISO Abbreviation:  J. Bacteriol.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-07-30     Completed Date:  2012-10-16     Revised Date:  2013-07-08    
Medline Journal Info:
Nlm Unique ID:  2985120R     Medline TA:  J Bacteriol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4169-77     Citation Subset:  IM    
Affiliation:
Department Isotope Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / metabolism*
Biosynthetic Pathways
Carbon Isotopes / metabolism
Chloroflexi / metabolism*
Gas Chromatography-Mass Spectrometry / methods
Isotope Labeling / methods
Spectrometry, Mass, Electrospray Ionization / methods
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Carbon Isotopes
Comments/Corrections

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