Division of Geological and Planetary Sciences, California Institute of Technology, 1200 E California Blvd, Pasadena, CA, 91125, USA.
Rapid Commun Mass Spectrom. 2021 Feb 28;35(4):e9007. doi: 10.1002/rcm.9007.
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.
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 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.
On-column focusing of SO 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 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 S values of cysteine and methionine that can be connected to biosynthetic pathways.
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.
以前，由于对有机含硫分子进行预处理化学和分析需要大量样本的要求具有挑战性，因此一直未能对含硫氨基酸（半胱氨酸和蛋氨酸）的天然丰度硫同位素组成进行分析。尽管在生物医药学、生态学、海洋学、生物地球化学和其他领域具有潜在的应用价值，但含硫氨基酸（半胱氨酸和蛋氨酸）的天然丰度硫同位素组成尚未得到研究。
半胱氨酸和蛋氨酸经热酸水解，然后用过甲酸定量氧化，生成半胱氨酸磺酸和蛋氨酸砜。这些稳定的氧化产物通过反相高效液相色谱（HPLC）分离，并通过离线液相色谱/质谱（LC/MS）进行验证。然后通过燃烧元素分析仪与同位素比质谱仪（EA/IRMS）测量纯化分析物的硫同位素比值（δ S 值）。EA 配备了温度升高的色谱柱和可编程氦载气流速。
在 EA/IRMS 系统中，SO 在柱上聚焦，洗脱过程中氦载气流速降低，大大提高了灵敏度，允许对 1 至 10 μg 硫进行精确（0.1-0.3‰ 1 s.d.）的 δ S 测量。我们验证了我们用于半胱氨酸和蛋氨酸纯化的方法不会对氨基酸和蛋白质标准品造成明显的分馏。对鱼类肌肉组织和细菌的概念验证测量表明，半胱氨酸和蛋氨酸的 δ S 值之间存在高达 4‰的差异，这可以与生物合成途径联系起来。
我们开发了一种灵敏、精确的方法，用于测量从生物样本中分离的半胱氨酸和蛋氨酸的天然丰度硫同位素组成。这种能力为氨基酸和蛋白质中的硫同位素开辟了广泛的应用，从用作生物体和环境中的示踪剂，到代谢和生物合成的基本方面。
