Abstract
We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with 13C, as a readily accessible source of multiple 13C-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)-13C-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 μM with excellent linearity for all of the calibration curves ( R2 ≥ 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-13C 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.
Highlights
Quantitative biology aims to explain the function of an entire biological system, and intracellular-metabolite analysis is an important aspect of this approach because it reveals functional information about the biochemical and physiological states of cells.[1,2] Metabolomics can provide “global” information on metabolites, but frequently this is not quantitative in nature and is difficult to apply in accurate modelings of intracellular processes required in systems biology and metabolic engineering
LC-MS methods have previously been reported for the simultaneous quantification of different classes of intracellular metabolites in microorganisms using mainly hydrophilic interaction liquid chromatography (HILIC)[3,4] and ion-pair reversed-phase chromatography.[5,6]
The optimized ZIC-pHILIC LC-HRMS method provided the quantitative analysis of a standard mixture of 74 metabolites of different classes and structurally related analogues simultaneously within a single 15 min analytical run compared with a similar 45 min method reported by others.[31,32]
Summary
Quantitative biology aims to explain the function of an entire biological system, and intracellular-metabolite analysis is an important aspect of this approach because it reveals functional information about the biochemical and physiological states of cells.[1,2] Metabolomics can provide “global” information on metabolites, but frequently this is not quantitative in nature (for example, by not providing precise concentrations) and is difficult to apply in accurate modelings of intracellular processes required in systems biology and metabolic engineering. Analyses employed by others (see Supporting Information Figure S-1).[15,33,34] The main advantages of the developed method are that it provides (1) rapid separation and confirmed identification of many key intracellular metabolites in 15 min, (2) improved annotation of metabolite peaks compared with other LC-MS methods, and (3) the convenience of using commercially available 13C spirulina over biosynthesized 13C IS.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
