Dynamic In Vivo Mapping of the Methylproteome Using a Chemoenzymatic Approach.

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Dynamic protein post-translational methylation is crucial for cellular functions like transcriptional regulation.
• Aberrant methylation is linked to diseases, including cancer, highlighting the need to catalog the methylproteome.
• Current methods for methylproteome analysis face limitations in enrichment and identification.

Purpose of the Study:

• To develop and validate a novel method for comprehensive methylproteome analysis.
• To identify known and novel protein propargylation sites using an l-methionine analogue.
• To demonstrate the in vivo application of the method for methylproteome profiling across organ systems.

Main Methods:

• Utilized ProSeMet, an l-methionine analogue, chemoenzymatically converted to ProSeAM for biorthogonal alkyne tagging of proteins.
• Employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) for direct detection and identification of tagged proteins.
• Applied selective enrichment strategies followed by LC-MS/MS for enhanced identification of methylated proteins and sites.

Main Results:

• Identified known and novel lysine, histidine, and arginine propargylation sites with high resolution on key proteins like HSPA8, eEF1A1, and PGAM1.
• Characterized 486 known and 221 novel protein propargylation sites across diverse cellular functions using enrichment methods.
• Demonstrated systemic ProSeMet delivery in mice, achieving protein propargylation across organ systems, including blood-brain barrier penetrance, and confirmed in vivo site-specific identification.

Conclusions:

• The ProSeMet-based pipeline offers a powerful approach for comprehensive methylproteome cataloging.
• This method enables site-specific identification of protein methylation, both in vitro and in vivo.
• The developed pipeline has broad applications for understanding methylproteome dynamics in health and disease contexts, particularly cancer.