Rapid and high coverage profile of human cysteineome

Cysteines play important roles in many biological activities, including protein folding, aging, signaling and etc. To identify and quantitatively profile the oxidation and reactivity of cysteines, cysteine-reactive compounds have been applied to covalently label and enrich the cysteines. And with mass spectrometry (MS), thousands of cysteines can be profiled at the same time with decoration of functional groups. However, with all the technology and methods developed to characterize cysteines, current datasets have only uncovered about 5% of the total cysteinome. Here we show a comprehensive and high coverage profile of more than 30,000 cysteines. With incorporation of new techniques - single-pot, solid phase-enhanced sample-preparation (SP3) and High-field asymmetric waveform ion mobility spectrometry (FAIMS), we have successfully identified as many as 14,306 cystienes in a single-shot experiment. With cysteine profiles in different cell lines, proteases and fractionations, our results have successfully expanded the cysteine library to a great extent.

半胱氨酸（Cysteine）在诸多生物学过程中发挥关键作用，涵盖蛋白质折叠、衰老、信号传导等领域。为实现对半胱氨酸氧化状态与反应活性的鉴定及定量分析，科研人员已采用半胱氨酸反应性化合物对其进行共价标记与富集。结合官能团修饰策略，借助质谱（Mass Spectrometry, MS）技术可同时对数千个半胱氨酸进行表征分析。然而，尽管目前已开发出多种半胱氨酸表征技术与方法，现有数据集仅覆盖了全部半胱氨酸组（cysteinome）的约5%。本研究构建了覆盖超过30000个半胱氨酸的全面高覆盖度表征图谱。通过引入两项新技术——单罐固相增强样品制备（single-pot, solid phase-enhanced sample-preparation, SP3）与高场不对称波形离子迁移谱（High-field asymmetric waveform ion mobility spectrometry, FAIMS），本研究在单次实验中成功鉴定出多达14306个半胱氨酸。结合不同细胞系、蛋白酶处理及组分分级后的半胱氨酸表征数据，本研究成果大幅拓展了半胱氨酸数据库的规模。