Protein lactylation is an emerging field. To advance the exploration of its biological functions, here we develop a comprehensive workflow that integrates proteomics to identify lactylated sites, genetic code expansion (GCE) for the expression of site-specifically lactylated proteins in living cells, and an integrated functional analysis (IFA) platform to evaluate their biological effects. Using a combined wet-and-dry-lab proteomics strategy, we identify a conserved lactylation at ALDOA-K147, which we hypothesize plays a significant biological role. Expression of this site-specifically lactylated ALDOA in mammalian cells reveals that this modification not only inhibits enzymatic activity but also induces gain-of-function effects. These effects reshaped ALDOA functionality by enhancing protein stability, promoting nuclear translocation, regulating adhesion-related gene expression, altering cell morphology and modulating ALDOA-interacting proteins. Our findings highlight the utility of the GCE-based workflow in establishing causal relationships between specific lactylation events and both target-specific and cell-wide changes, advancing our understanding of protein lactylation's functional impact.
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