Confident Identification and Quantification of Mouse Brain Tissues Reveals Sirtuin 5-Dependent Regulation

Area of Science:

• Biochemistry
• Proteomics
• Neuroscience

Background:

• Lysine methylmalonylation is a post-translational modification linked to mitochondrial dysfunction.
• Its analysis is challenging due to isobaric overlap with succinylation, hindering broader study.
• Previous research has been limited, leaving its distribution and function largely unexplored.

Purpose of the Study:

• To develop a robust proteomic workflow for confident identification and quantification of lysine methylmalonylation.
• To investigate the methylmalonylome in mouse brain tissue, particularly in relation to Sirtuin-5 (SIRT5) activity.
• To explore the functional consequences of methylmalonylation on protein function, specifically myelin basic protein (MBP).

Main Methods:

• Developed a workflow combining antibody-based enrichment with data-independent acquisition mass spectrometry (DIA-MS).
• Utilized synthetic peptide standards to define distinguishing analytical features for isobaric modifications.
• Applied the workflow to Sirtuin-5 (SIRT5) knockout and wild-type mouse brain tissues.

Main Results:

• Successfully identified 44 methylmalonylated peptides across 41 proteins in mouse brain.
• Identified enrichment of methylmalonylated proteins in neuronal, myelin-associated, and mitochondrial pathways.
• Demonstrated that methylmalonylation of MBP impairs its lipid-binding capacity, affecting myelin stability.
• Observed increased methylmalonylation sites in SIRT5-deficient brains, suggesting SIRT5-mediated regulation.

Conclusions:

• The developed workflow enables confident identification of lysine methylmalonylation, overcoming isobaric interference.
• Methylmalonylation is a widespread and regulated post-translational modification in the brain.
• This modification plays a role in myelin stability and provides a framework for studying metabolically driven protein acylation in neurological contexts.