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RationaleSulfur 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. MethodsCysteine 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. ResultsOn‐column focusing of SO2in 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. ConclusionsWe 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.
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This content will become publicly available on December 24, 2025
Order of amino acid recruitment into the genetic code resolved by last universal common ancestor’s protein domains
The current “consensus” order in which amino acids were added to the genetic code is based on potentially biased criteria, such as the absence of sulfur-containing amino acids from the Urey–Miller experiment which lacked sulfur. More broadly, abiotic abundance might not reflect biotic abundance in the organisms in which the genetic code evolved. Here, we instead identify which protein domains date to the last universal common ancestor (LUCA) and then infer the order of recruitment from deviations of their ancestrally reconstructed amino acid frequencies from the still-ancient post-LUCA controls. We find that smaller amino acids were added to the code earlier, with no additional predictive power in the previous consensus order. Metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Methionine and histidine were added to the code earlier than expected from their molecular weights and glutamine later. Early methionine availability is compatible with inferred early use of S-adenosylmethionine and early histidine with its purine-like structure and the demand for metal binding. Even more ancient protein sequences—those that had already diversified into multiple distinct copies prior to LUCA—have significantly higher frequencies of aromatic amino acids (tryptophan, tyrosine, phenylalanine, and histidine) and lower frequencies of valine and glutamic acid than single-copy LUCA sequences. If at least some of these sequences predate the current code, then their distinct enrichment patterns provide hints about earlier, alternative genetic codes.
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- Award ID(s):
- 2333243
- PAR ID:
- 10621340
- Publisher / Repository:
- National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 121
- Issue:
- 52
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- e2410311121
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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