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

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.