Practical considerations for amino acid isotope analysis

Silverman, Shaelyn N.; Phillips, Alexandra A.; Weiss, Gabriella M.; Wilkes, Elise B.; Eiler, John M.; Sessions, Alex L.

doi:10.1016/j.orggeochem.2021.104345

Cited by 27 publications

(35 citation statements)

References 252 publications

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“…Although GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS have their own strength and weakness, which is of critical importance for determination of AA δ 15 N values (Silverman et al 2022), they have not been intercalibrated yet. To address this issue, we aimed to systematically compare δ 15 N Glu , δ 15 N Phe , and TP values, three major outcomes for biogeochemical CSIA‐AA studies, among the three analytical methods using biological samples containing matrices.…”

mentioning

confidence: 99%

Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

Ishikawa

Ogawa

Sun

et al. 2022

Limnology & Ocean Methods

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Compound-specific isotope analysis of nitrogen (δ 15 N) in amino acids (CSIA-AA) has significantly contributed to environmental sciences such as anthropology, biogeochemistry, and ecology. Several methods exist for determining δ 15 N of amino acids (AAs). Although these methods have their own strength and weakness, they have not been intercalibrated yet, especially for biological samples with matrices. To address this issue, we systematically compared AA δ 15 N values among three methods using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), preparative liquid chromatography (LC) separation followed by elemental analyzer/ IRMS (LC Â EA/IRMS), and LC separation followed by GC/C/IRMS (LC Â GC/C/IRMS). The δ 15 N values of glutamic acid (δ 15 N Glu ) and phenylalanine (δ 15 N Phe ) in fish muscle, two crucial AAs for estimating the trophic positions (TPs) of organisms, were compared among methods. Although a significant difference in fish muscle δ 15 N Glu values was found among the three analytical methods, their δ 15 N Glu and δ 15 N Phe values were fairly consistent between all pairs of methods (n = 8, R 2 = 0.9968 for GC/C/IRMS vs. LC Â GC/C/IRMS; 0.9936 for LC Â EA/IRMS vs. LC Â GC/C/IRMS; and 0.9912 for GC/C/IRMS vs. LC Â EA/IRMS), which resulted in similar TP estimates among the methods. Thus, the results provide empirical validation that the CSIA-AA is comparable among different methods in interdisciplinary research fields. We also highlighted some critical features of each of the three analytical methods that can be used as a guideline for future CSIA-AA research.

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mentioning

confidence: 99%

Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

Ishikawa

Ogawa

Sun

et al. 2022

Limnology & Ocean Methods

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“…36,37 A subset of these samples, representing dried, powdered samples of aboveground tissues grown under highly elevated (2255 ± 148 ppmv), elevated (733 ± 18 ppmv), or subambient (322 ± 4 ppmv) pCO 2 levels and ambient pO 2 (ca 21%) were selected for the present study to include conditions that should foster different balances of photorespiration (O 2 fixation) and photosynthesis (CO 2 fixation), although these fluxes were not quantified. A total of 10-15 mg of the powdered samples, combined from two or three biological replicates, was weighed into 40 mL VOA vials with Teflon-lined caps and then subjected to acid hydrolysis (6 N HCl, 16 h, 108 C, argon headspace), with conditions selected to optimize recovery of serine (reviewed in Silverman et al 38 ). This type of acid hydrolysis has been shown to have little impact on serine's carbon isotopic composition in prior studies, 39,40 and was confirmed for our specific protocol by subjecting an external standard containing SERC0 to the entire preparatory workflow.…”

Section: Plant Sample Preparationmentioning

confidence: 99%

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

Wilkes

Sessions

Zeichner

et al. 2022

Rapid Comm Mass Spectrometry

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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.

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“…The sulfur isotopic composition of DOS SPE indicates the relative balance of these two sources. organic compounds (29)(30)(31) and should be negligible for sulfur atoms that do not participate in hydrophobic binding interactions. For example, hydrophobic separation of volatile organic sulfur compounds by gas chromatography does not yield fractionation of sulfur isotopes across peaks (32).…”

Section: Significancementioning

confidence: 99%

Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter

Phillips

White

Seidel

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Marine dissolved organic matter (DOM) is a major reservoir that links global carbon, nitrogen, and phosphorus. DOM is also important for marine sulfur biogeochemistry as the largest water column reservoir of organic sulfur. Dissolved organic sulfur (DOS) can originate from phytoplankton-derived biomolecules in the surface ocean or from abiotically “sulfurized” organic matter diffusing from sulfidic sediments. These sources differ in 34 S/ 32 S isotope ratios (δ 34 S values), with phytoplankton-produced DOS tracking marine sulfate (21‰) and sulfurized DOS mirroring sedimentary porewater sulfide (∼0 to –10‰). We measured the δ 34 S values of solid-phase extracted (SPE) DOM from marine water columns and porewater from sulfidic sediments. Marine DOM SPE δ 34 S values ranged from 14.9‰ to 19.9‰ and C:S ratios from 153 to 303, with lower δ 34 S values corresponding to higher C:S ratios. Marine DOM SPE samples showed consistent trends with depth: δ 34 S values decreased, C:S ratios increased, and δ 13 C values were constant. Porewater DOM SPE was 34 S-depleted (∼-0.6‰) and sulfur-rich (C:S ∼37) compared with water column samples. We interpret these trends as reflecting at most 20% (and on average ∼8%) contribution of abiotic sulfurized sources to marine DOS SPE and conclude that sulfurized porewater is not a main component of oceanic DOS and DOM. We hypothesize that heterogeneity in δ 34 S values and C:S ratios reflects the combination of sulfurized porewater inputs and preferential microbial scavenging of sulfur relative to carbon without isotope fractionation. Our findings strengthen links between oceanic sulfur and carbon cycling, supporting a realization that organic sulfur, not just sulfate, is important to marine biogeochemistry.

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Practical considerations for amino acid isotope analysis

Cited by 27 publications

References 252 publications

Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

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

Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter

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