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

Abstract

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. 1 | INTRODUCTION The sulfur isotopic compositions of amino acids (AAs) are virtually unexplored but may hold significant utility across diverse scientific disciplines. In biomedicine, pilot studies have suggested that cysteine and methionine δ 34 S values could indicate disease progression as sulfur metabolism is dysregulated at the onset of liver cancer. 1 In archeology, bulk protein δ 34 S values of mummy hair 2 and mammalian collagen 3 have been used to reconstruct ancestral migration and reliance on fish protein, indicating this as a promising direction for

Rapid, Concurrent Formation of Organic Sulfur and Iron Sulfides During Experimental Sulfurization of Sinking Marine Particles

“…A large proportion (42–65%) of the assimilatory S in microplankton is typically found as proteins and polypeptides (Cuhel et al., 1982), specifically the amino acids cysteine, which is a thiol, and methionine, which is an organic monosulfide. Cysteine and methionine are highly susceptible to oxidation, both in the environment and during laboratory handling, which will produce sulfoxide (Vogt, 1995) and/or sulfonate (Phillips et al., 2021). The AVS hydrolysis method used here to isolate OM Res is less intense (shorter duration and lower temperature) but otherwise similar to some early methods for protein hydrolysis (e.g., 24 hr, 110°, 6N HCl) (Hill, 1965).…”

Rapid, concurrent formation of organic sulfur and iron sulfides during experimental sulfurization of sinking marine particles

“…Cysteine and methionine are highly susceptible to oxidation, both in the environment and during laboratory handling, which will produce sulfoxide (Vogt, 1995) and/or sulfonate (Phillips et al, 2021). The AVS hydrolysis method used here to isolate OMRes is less intense (shorter duration and lower temperature) but otherwise similar to some early methods for protein hydrolysis (e.g., 24 hrs, 110°, 6N HCl) (Hill, 1965).…”

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

“…Nevertheless, these techniques are still in early stages of development with limited application thus far to samples of interest to biogeochemists and ecologists. For example, it is challenging to isolate methionine from natural samples, 27 and the measurements of meteoritic alanine had propagated uncertainties for individual carbon positions that could not differentiate natural variations on Earth.…”