Presenter Bio: I am a PhD student in Biochemistry at McGill University. I use structural and biophysical techniques to study PRL phosphatases, and I am currently developing an LC-MS/MS–based method to detect cysteine phosphorylation in cells.
Relevant Financial Disclosures
(within past 24 months, reported on Jul 14, 2025)
No relevant financial relationship(s) to disclose.
Abstract
INTRODUCTION:
Cysteine phosphorylation in proteins plays a regulatory role in signaling but remains underrepresented in phosphoproteomics due to its transient and chemically labile nature. Phosphatases of regenerating liver (PRLs) are cystine-based phosphatases that form a stable phosphocysteine intermediate during catalysis, which can accumulate endogenously in cells. All three human PRL isoforms are involved in cell growth and magnesium homeostasis and are frequently associated with cancer metastasis. Although both their catalytic and non-catalytic functions have been implicated in promoting metastasis, the underlying mechanism remains poorly understood. Quantifying phosphocysteine levels in cells and tissues may provide insight into their role in tumor progression.
OBJECTIVES:
The absence of PRL isoform-specific antibodies, combined with the acid- and heat-labile nature of phosphocysteine, has limited its characterization in biological systems. This study aims to establish a derivatization-based mass spectrometry workflow for the quantitative detection of PRL cysteine phosphorylation. A broader goal is to determine whether other cellular proteins also harbor stable cysteine phosphorylation.
METHODS:
Cell lysates or purified protein samples were first denatured, and free cysteine residues were reduced and alkylated to prevent nonspecific labeling. Phosphocysteine was then hydrolyzed by controlled heating, converting it to reactive thiols. The newly exposed thiols were selectively labeled with maleimide-based reagents. Labeled proteins were enriched using affinity purification, followed by tryptic digestion. Phosphocysteine-derived peptides were identified and quantified by mass spectrometry.
RESULTS:
A robust workflow was developed for detecting cysteine phosphorylation in recombinant PRLs under denaturing conditions. Denaturation in guanidine hydrochloride rendered the phosphocysteine intermediate more resistant to premature hydrolysis, enabling its preservation throughout the initial steps of the workflow. Following thermal hydrolysis, selective labeling of phosphocysteine-derived cysteines was achieved using biotin-maleimide. Labeled peptides from recombinant PRL2 expressed in E. coli were reliably detected by both MALDI-TOF and LC-MS/MS. NeutrAvidin-based enrichment of biotinylated proteins significantly enhanced detection sensitivity, allowing confident identification of modified peptides over background. Systematic optimization of reduction, alkylation, and labeling steps minimized nonspecific reactivity and improved recovery of phosphocysteine-derived peptides.
CONCLUSION:
This workflow offers a reliable, MS-compatible approach for the selective detection of cysteine phosphorylation in PRLs and is adaptable to other phosphocysteine-containing proteins. Future work will apply this strategy to mammalian cells and tissues to evaluate phosphocysteine levels in PRLs and explore their contribution to metastatic cancer.
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
