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

Abstract

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.

Oxygen Isotope Analysis of Nanomole Phosphate Using PO3– Fragment in ESI-Orbitrap-MS

“…Sulfur isotope analysis on carbonyl sulfide (COS or OCS) has been done using S + fragment ions to obviate the need for tedious chemical transformations . Fragmentation techniques on the Orbitrap-MS coupled with ESI or electron impact ionization (EI) source have been used to measure the position-specific carbon isotope composition of terrestrial and extraterrestrial amino acids, including methionine, serine, and alanine. − In this study, we demonstrated that the use of fragmentation methods permits the interference-free, high-precision isotope analysis of phosphate. We anticipate that the fragmentation method can be further explored to enhance the sensitivity and accuracy of isotope analysis for oxyanions.…”

Comparison of Carbon Isotope Ratio Measurement of the Vanillin Methoxy Group by GC-IRMS and 13C-qNMR

“…The 13 C-qNMR used in carbon isotope ratio measurement is also known as irm- 13 C NMR: isotope ratio measured by NMR, and as SNIF-NMR: Site-Specific Natural Isotope Fractionation determined by NMR . Recently, the high-resolution Orbitrap-MS was also used to measure site-specific carbon isotopic composition in serine . Thus, the 13 C- qNMR and Orbitrap-MS direct access to site-specific isotope compositions of compounds at natural abundance provides much more information for classification and/or discrimination of molecules as a function of their geographical and/or chemical origin, especially in food products. ,, …”

“…2 Recently, the high-resolution Orbitrap-MS was also used to measure sitespecific carbon isotopic composition in serine. 3 Thus, the 13 C-qNMR and Orbitrap-MS direct access to site-specific isotope compositions of compounds at natural abundance provides much more information for classification and/or discrimination of molecules as a function of their geographical and/or chemical origin, especially in food products. 2,4,5 However, applying IRMS for site-specific determination of carbon isotope ratios of the methoxy group in a given molecule is also possible, and this offers a means to validate the 13 C-qNMR measurements.…”

Natural Abundance Isotope Ratio Measurements of Organic Molecules Using 21 T FTICR MS

“…A general characterization of Orbitrap performance at measuring isotopic patterns accurately was also conducted . More recently, investigations have demonstrated methods for online GC-FTMS for compound and site-specific isotope measurements . These methods have generally been limited to low molecular weight species, partly due to the limited mass resolution and charge capacity of Orbitrap–noting, that to accurately measure the abundance of isotopologues, you must be able to detect and resolve them.…”

“…20 More recently, investigations have demonstrated methods for online GC-FTMS for compound 21 and site-specific isotope measurements. 22 These methods have generally been limited to low molecular weight species, partly due to the limited mass resolution and charge capacity of Orbitrap−noting, that to accurately measure the abundance of isotopologues, you must be able to detect and resolve them. Limited mass resolving power and charge capacity preclude this for larger, complex molecules.…”