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Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism

Wilkes, Elise B. and Sessions, Alex L. and Zeichner, Sarah S. and Dallas, Brooke and Schubert, Brian and Jahren, A. Hope and Eiler, John M. (2022) Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism. Rapid Communications in Mass Spectrometry, 36 (18). Art. No. e9347. ISSN 0951-4198. doi:10.1002/rcm.9347.

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Rationale: Position-specific ¹³C/¹²C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. Methods: Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The ¹³C/¹²C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ¹³C values relative to a working standard. Results: This methodology constrains position-specific δ¹³C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ¹³C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7–5‰ for each carbon position. Position-specific δ¹³C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO₂, selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. Conclusions: This study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

Item Type:Article
Related URLs:
URLURL TypeDescription Information
Wilkes, Elise B.0000-0002-3379-7832
Sessions, Alex L.0000-0001-6120-2763
Zeichner, Sarah S.0000-0001-8897-7657
Dallas, Brooke0000-0002-1313-3270
Schubert, Brian0000-0001-9459-2059
Jahren, A. Hope0000-0003-3884-1676
Eiler, John M.0000-0001-5768-7593
Additional Information:© 2022 John Wiley & Sons Ltd. Issue Online: 21 July 2022; Version of Record online: 21 July 2022; Accepted manuscript online: 29 June 2022; Manuscript accepted: 23 June 2022; Manuscript revised: 22 June 2022; Manuscript received:16 December 2021. Research Funding: Agouron Institute. Grant Number: AI-F-GB54.19.2. Caltech Center for Environmental and Microbial Interactions. Department of Energy. Grant Number: DE-SC0016561. NASA Astrobiology Institute. Grant Number: 80NSSC18M094. National Science Foundation Geobiology. Grant Number: EAR-1921330. Research Council of Norway. Grant Number: 223272. Simons Foundation. Peer Review: The peer review history for this article is available at Data Availability Statement: Data available on request from the authors.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Agouron InstituteAI-F-GB54.19.2
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Department of Energy (DOE)DE-SC0016561
Research Council of Norway223272
Simons FoundationUNSPECIFIED
Issue or Number:18
Record Number:CaltechAUTHORS:20220708-948968900
Persistent URL:
Official Citation:Wilkes, EB, Sessions, AL, Zeichner, SS, et al. Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism. Rapid Commun Mass Spectrom. 2022; 36(18):e9347. doi:10.1002/rcm.9347
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115439
Deposited By: Tony Diaz
Deposited On:11 Jul 2022 18:12
Last Modified:29 Sep 2022 21:23

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