Compound Specific Sulfur Isotopes of the Amino Acids Cysteine and Methionine

Compound specific amino acid isotope measurements are an emerging geochemistry tool, informing diverse topics such as biochemical pathways, trophic dynamics, nutrient cycling, and disease biomarkers. However, despite recent advancements in methodology and instrumentation, little work has focused on the sulfur containing amino acids; cysteine and methionine. A combination of high sulfur backgrounds and uncontrollable reactivity of sulfur species have hindered these analyses. This study presents progress towards the first δ³⁴S measurements of sulfur-containing amino acids. The method entails acid hydrolysis of protein standards and natural samples. Next, cysteine is quantitatively oxidized to cysteic acid and methionine to methionine sulfone. These more stable oxidized species are then separated by HPLC, verified by LCMS, and measured by EA-IRMS. The new Thermo EA-IRMS Isolink has allowed detection of less than 50 nanomoles of organic sulfur with 0.3‰ precision. Amino acid standards and protein standards were used to test the method. Cysteine standards have a δ³⁴S value of 5.81‰ (± 0.19). After running this standard throughout the method, the isotope value was within error of its starting value (5.55‰ ± 0.19). Methionine was similarly unchanged throughout the procedure, beginning at 7.44‰ ± 0.19 and ending at 7.56‰ ± 0.19. These results with standards partially validate the method, ensuring no measurable fractionation has been introduced. Isotope mass balance with a model protein, bovine serum albumin, was also demonstrated. In bacteria, cysteine is often the source of sulfur for methionine synthesis. This pathway has not been studied for sulfur isotope effects, but it is reasonable to assume normal KIEs leading to enrichment of cysteine and depletion of methionine. Initial measurements of E. Coli and Pseudomonas display this pattern, with cysteine δ³⁴S values of 1.59‰ and 1.13‰, and methionine values of 0.27‰ and -0.07‰, respectively. Errors on theses measurement are reported as ± 0.10‰, the standard deviation of standards run in triplicate. With heterogeneity demonstrated in a predictable direction, there is precedent to continue a survey of microbial biomass. At AGU, further measurements will be presented from culturing studies, as well as progress towards natural samples such as keratin and plants.