Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry
Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry
复制标题
化学衍生化-液相色谱-质谱法灵敏分析单个人宫颈癌细胞中的多种低分子量硫醇
作者：
Xiao Hua-Ming;Wang Xian;Liao Quan-Lan;Zhao Shuai;Huang Wei-Hua;Feng Yu-Qi
低分子量硫醇是一类重要的小分子化合物，在生理和病理过程中起着调节或维持氧化还原稳态的作用。评估生物系统中低分子量硫醇的浓度可以提供有关生理过程和一些疾病的早期诊断的有价值的信息。在这里，我们开发了一种通过化学衍生辅助液相色谱-质谱法（LC-MS）同时测定单细胞中多种LWM硫醇浓度的方法。在此方法中，我们合成了一对稳定同位素试剂N-（吖啶-9-基）-2-溴乙酰胺（AYBA）和N-（1，2，3，4-[2 H4]-吖啶-9-基）-2-溴乙酰胺（[2 H4]AYBA）。AYBA用于衍生人宫颈癌（HeLa）细胞中的LWM硫醇，而[2 H4]AYBA用于衍生标准LWM硫醇，以制备用于LC-MS方法开发的内标物。AYBA衍生化大大提高了LC-MS对LWM硫醇的检测灵敏度，从而实现了LC-MS对1000个HeLa细胞中多种LWM硫醇的同时检测。最后，将该方法成功地应用于单个HeLa细胞中多种LWM硫醇的定量分析，以及单个HeLa细胞中LWM硫醇在氧化应激处理前后的含量变化。因此，检测到六种LMW硫醇，包括半胱胺、半胱氨酸、谷胱甘肽、高半胱氨酸、硫化氢和泛酰巯基乙胺。
Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.