PMID- 29533656
OWN - NLM
STAT- MEDLINE
DCOM- 20190415
LR  - 20190415
IS  - 1520-6882 (Electronic)
IS  - 0003-2700 (Linking)
VI  - 90
IP  - 7
DP  - 2018 Apr 3
TI  - Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of 
      Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 
      (13)C-Labeled Standards Derived from Spirulina.
PG  - 4470-4477
LID - 10.1021/acs.analchem.7b04758 [doi]
AB  - We have investigated the applicability of commercially available lyophilized 
      spirulina ( Arthrospira platensis), a microorganism uniformly labeled with (13)C, 
      as a readily accessible source of multiple (13)C-labeled metabolites suitable as 
      internal standards for the quantitative determination of intracellular bacterial 
      metabolites. Metabolites of interest were analyzed by hydrophilic-interaction 
      liquid chromatography coupled with high-resolution mass spectrometry. Multiple 
      internal standards obtained from uniformly (U)-(13)C-labeled extracts from 
      spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the 
      identification and quantification of intracellular metabolites. Extraction of the 
      intracellular metabolites of Clostridium autoethanogenum using 2:1:1 
      chloroform/methanol/water was found to be the optimal method in comparison with 
      freeze-thaw, homogenization, and sonication methods. The limits of quantification 
      were </=1 muM with excellent linearity for all of the calibration curves ( R(2) >/= 
      0.99) for 74 metabolites. The precision and accuracy were found to be within 
      relative standard deviations (RSDs) of 15% for 49 of the metabolites and within 
      RSDs of 20% for all of the metabolites. The method was applied to study the 
      effects of feeding different levels of carbon monoxide (as a carbon source) on 
      the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in 
      continuous culture over 35 days. Using LC-IDMS with U-(13)C spirulina allowed the 
      successful quantification of 52 metabolites in the samples, including amino 
      acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method 
      provided absolute quantitative data on intracellular metabolites that was 
      suitable for computational modeling to understand and optimize the C. 
      autoethanogenum metabolic pathways active in gas fermentation.
FAU - Schatschneider, Sarah
AU  - Schatschneider S
AD  - Centre for Analytical Bioscience, School of Pharmacy , University of Nottingham , 
      Nottingham NG7 2RD , U.K.
FAU - Abdelrazig, Salah
AU  - Abdelrazig S
AUID- ORCID: 0000-0001-6231-1267
AD  - Centre for Analytical Bioscience, School of Pharmacy , University of Nottingham , 
      Nottingham NG7 2RD , U.K.
FAU - Safo, Laudina
AU  - Safo L
AD  - Centre for Analytical Bioscience, School of Pharmacy , University of Nottingham , 
      Nottingham NG7 2RD , U.K.
FAU - Henstra, Anne M
AU  - Henstra AM
AD  - Clostridia Research Group, SBRC-Nottingham, a BBSRC/EPSRC Synthetic Biology 
      Research Centre, School of Life Sciences , University of Nottingham , Nottingham 
      NG7 2RD , U.K.
FAU - Millat, Thomas
AU  - Millat T
AD  - Clostridia Research Group, SBRC-Nottingham, a BBSRC/EPSRC Synthetic Biology 
      Research Centre, School of Life Sciences , University of Nottingham , Nottingham 
      NG7 2RD , U.K.
FAU - Kim, Dong-Hyun
AU  - Kim DH
AD  - Centre for Analytical Bioscience, School of Pharmacy , University of Nottingham , 
      Nottingham NG7 2RD , U.K.
FAU - Winzer, Klaus
AU  - Winzer K
AD  - Clostridia Research Group, SBRC-Nottingham, a BBSRC/EPSRC Synthetic Biology 
      Research Centre, School of Life Sciences , University of Nottingham , Nottingham 
      NG7 2RD , U.K.
FAU - Minton, Nigel P
AU  - Minton NP
AD  - Clostridia Research Group, SBRC-Nottingham, a BBSRC/EPSRC Synthetic Biology 
      Research Centre, School of Life Sciences , University of Nottingham , Nottingham 
      NG7 2RD , U.K.
FAU - Barrett, David A
AU  - Barrett DA
AD  - Centre for Analytical Bioscience, School of Pharmacy , University of Nottingham , 
      Nottingham NG7 2RD , U.K.
LA  - eng
GR  - BB/K00283X/1/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20180321
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
RN  - 0 (Carbon Isotopes)
SB  - IM
MH  - Carbon Isotopes
MH  - Chromatography, Liquid
MH  - Clostridium/cytology/*metabolism
MH  - Hydrophobic and Hydrophilic Interactions
MH  - *Indicator Dilution Techniques
MH  - Mass Spectrometry
MH  - Spirulina/*metabolism
EDAT- 2018/03/14 06:00
MHDA- 2019/04/16 06:00
CRDT- 2018/03/14 06:00
PHST- 2018/03/14 06:00 [pubmed]
PHST- 2019/04/16 06:00 [medline]
PHST- 2018/03/14 06:00 [entrez]
AID - 10.1021/acs.analchem.7b04758 [doi]
PST - ppublish
SO  - Anal Chem. 2018 Apr 3;90(7):4470-4477. doi: 10.1021/acs.analchem.7b04758. Epub 
      2018 Mar 21.