Chemical-proteomic strategies to study cysteine posttranslational modifications

Abstract

Cysteine residues on proteins play important catalytic and regulatory roles in complex proteomes. These functional residues can be modified under physiological conditions by posttranslational modifications (PTMs) to regulate protein activities and modulate cysteine reactivity. Many PTMs are highly labile and dynamic, rendering it difficult to detect modified proteins within complex systems. To contribute to the chemical-proteomic methods currently available, chemical probe-Mass Spectrometry (MS) platforms were developed to study oxidative cysteine modifications. A MS platform for the assessment of S-nitrosation in vitro identified Cys329 of Cathepsin D (CTSD) as highly sensitive to S-nitrosothiol formation. To achieve a more physiological relevant representation of S-nitrosation, this platform was later adapted for study in live cells using a caged electrophile, Caged BK. Additionally, oscillation of cysteine oxidation as a function of circadian rhythm in Drosophila melanogaster and human samples was explored. As a compliment to these MS platforms, a 4-aminopiperidine-based cysteine-reactive probe library was developed. These probes have been used to target specific reactive cysteines as an alternate way to regulate protein function and can be used as tools to provide insight into the roles of these residues in protein activities.