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Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry
Author(s) -
Phillips Alexandra A.,
Wu Fenfang,
Sessions Alex L.
Publication year - 2021
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.9007
Subject(s) - chemistry , cysteic acid , chromatography , sulfur , mass spectrometry , methionine , isotope ratio mass spectrometry , cysteine , amino acid , organic chemistry , biochemistry , cystine , enzyme
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 (δ 34 S 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 SO 2 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.) δ 34 S 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 δ 34 S 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.