Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry
Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry
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DOI：
10.1002/rcm.9007
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发表时间：
2021-02-28
影响因子：
2
通讯作者：
Sessions, Alex L.
中科院分区：
文献类型：
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作者：
Phillips, Alexandra A.;Wu, Fenfang;Sessions, Alex L.
有机含硫分子的硫同位素分析以前受到具有挑战性的制备化学和大样品量分析要求的阻碍。天然丰度的硫同位素组成的含硫氨基酸，半胱氨酸和蛋氨酸，因此还没有被调查，尽管在生物医学，生态学，海洋学，地球化学，和其他领域的潜在效用。方法半胱氨酸和蛋氨酸进行热酸水解，然后在过甲酸定量氧化，得到磺基丙氨酸和蛋氨酸砜。然后通过反相高效液相色谱（HPLC）分离这些稳定的氧化产物，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耦合同位素比质谱法（EA/IRMS）测量纯化分析物的硫同位素比（Δ S-34值）。结果EA/IRMS系统中SO2的柱上聚焦，结合洗脱过程中减少的He载流，大大提高了灵敏度，使其精确度（0.1-0.3 ppm）达到1s.d.δ S-34测量值为1至10 μ g硫。我们验证了我们用于纯化半胱氨酸和蛋氨酸的方法是使用氨基酸和蛋白质标准品进行可忽略不计的分级。鱼肌肉组织和细菌的概念验证测量表明，差异高达4千分之一之间的三角洲S-34值的半胱氨酸和蛋氨酸，可以连接到biosynthetic pathways.Conclusions我们已经开发出一种灵敏，精确的方法来测量天然丰度的硫同位素组成的半胱氨酸和蛋氨酸分离的生物样品。这种能力开辟了硫同位素在氨基酸和蛋白质中的多种应用，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。
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 (delta S-34 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 SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1-0.3 parts per thousand 1 s.d.) delta S-34 measurements of 1 to 10 mu g 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 parts per thousand between the delta S-34 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.