Subcellular Proteomic Mapping of Lysine Lactylation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein lactylation is a key post-translational modification (PTM) influencing macrophage polarization, immune regulation, and tumor cell growth.
• Traditional methods for lactylation study often miss low-abundance lactylated peptides due to reliance on whole-cell lysates.

Purpose of the Study:

• To develop and apply a subcellular fractionation approach for comprehensive lactylation site mapping.
• To identify novel lactylated proteins and sites, particularly low-abundance ones, and explore their functional contexts.

Main Methods:

• Subcellular fractionation was used to isolate distinct cellular compartments.
• Mass spectrometry-based proteomics was employed to map lysine lactylation (Kla) sites within each fraction.
• Pathway enrichment analysis was performed on identified lactylated proteins.

Main Results:

• Identified 1,217 lysine lactylation (Kla) sites on 553 proteins across four subcellular fractions.
• Discovered 36 novel Kla proteins and 223 new Kla sites, including low-abundance ones.
• Pathway analysis showed distinct functions for lactylated proteins based on subcellular localization and suggested PTM crosstalk.

Conclusions:

• Subcellular fractionation is a powerful strategy for uncovering previously unidentified lactylation sites and proteins.
• This approach enhances the understanding of lactylation's role in diverse cellular pathways and its potential interplay with other PTMs.