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Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry

Phillips, Alexandra A. and Wu, Fenfang and Sessions, Alex L. (2021) Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 35 (4). Art. No. e9007. ISSN 0951-4198. doi:10.1002/rcm.9007.

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Rationale: Sulfur isotope analysis of organic sulfur‐containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural‐abundance sulfur isotopic compositions of the sulfur‐containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields. Methods: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed‐phase high‐performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ³⁴S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature‐ramped chromatographic column and programmable helium carrier flow rates. Results: On‐column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1–0.3‰ 1 s.d.) δ³⁴S measurements of 1 to 10 μg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof‐of‐concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ³⁴S values of cysteine and methionine that can be connected to biosynthetic pathways. Conclusions: We have developed a sensitive, precise method for measuring the natural‐abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis.

Item Type:Article
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Phillips, Alexandra A.0000-0001-5959-5238
Sessions, Alex L.0000-0001-6120-2763
Alternate Title:Compound‐specific sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope‐ratio mass spectrometry
Additional Information:© 2020 John Wiley & Sons. Issue Online: 07 January 2021; Version of Record online: 07 January 2021; Accepted manuscript online: 17 November 2020; Manuscript accepted: 14 November 2020; Manuscript revised: 11 November 2020; Manuscript received: 25 September 2020. The peer review history for this article is available at We would like to thank the technicians as well as the directors of the Proteomics Exploration Lab at Caltech: Sonja Hess and Anne Radimin for assistance with LC/MS operation and FDAA derivatization. This project benefited from the use of instrumentation made available by the Caltech Environmental Analysis Center and we acknowledge its director Nathan Dalleska for assistance with the HPLC-UV system. Caltech professors Victoria Orphan and Jess Adkins volunteered lab space for this project and we thank them and their lab technicians Stephanie Connon, Jared Markse, Grecia Lopez, and Guillaume Paris. We also thank high school interns Zekaria Beshir, Brenna Bowlen, and Hannah Betts for their valuable assistance on the project. We acknowledge Reto Wijker for providing cultures, media recipes, and assistance with microbial culturing. We thank colleagues for their valuable insight and review of early manuscripts: Ted Present, Preston Kemeny, Eryn Eitel, Hannah Dion-Kirschner, Frank Pavia, and Makayla Betts. We also thank Guillaume Tcherkez for procedures and advice on oxidation via MTO as well as Emilia Hernandez and Tony Wang for assistance testing oxidation of organics to sulfate. Tony Wang is also acknowledged for his help with optimizing initial EA/IRMS parameters. Funding for this project was provided by the NSF (Grant 1436566) and NASA Astrobiology Institute (Grant 80NSSC18M094).
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Subject Keywords:Amino Acids, Cysteine, Methionine, Sulfur Isotopes, EA/IRMS, CSIA
Issue or Number:4
Record Number:CaltechAUTHORS:20201117-132832420
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Official Citation:Phillips, AA, Wu, F, Sessions, AL. Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry. Rapid Commun Mass Spectrom. 2021; 35:e9007.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106708
Deposited By: George Porter
Deposited On:17 Nov 2020 22:11
Last Modified:16 Nov 2021 18:56

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