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.