Subcellular Proteomic\nMapping of Lysine Lactylation

Protein lactylation\nis a novel post-translational modification\n(PTM) involved in many important physiological processes such as macrophage\npolarization, immune regulation, and tumor cell growth. However, traditional\nmethodologies for studying lactylation have predominantly relied on\npeptide enrichment from whole-cell lysates, which tend to favor the\ndetection of high-abundance peptides, thus limiting the identification\nof low-abundance lactylated peptides. To address this limitation,\nhere, we employed subcellular fractionation to separate proteins and\nmap lactylated peptides from each isolated subcellular fraction using\na model cell line. In brief, we identified 1,217 lysine lactylation\n(Kla) sites on 553 proteins across four subcellular fractions. Subsequent\npathway enrichment analysis revealed that Kla proteins participate\nin distinct pathways depending on the subcellular contexts. In addition,\nthis subcellular fractionation method enabled the discovery of 36\npreviously unreported Kla proteins and 223 novel Kla sites, many of\nwhich are present in low abundance. Notably, several proteins contain\nmultiple newly identified Kla sites, exemplified by the transcriptional\nregulator ATRX. Furthermore, our results indicate the possibility\nof PTM crosstalk between Kla and other PTMs such as ubiquitination\nand sumoylation. In conclusion, subcellular fractionation facilitates\nthe identification of Kla proteins that have been previously uncovered\nand could be overlooked by affinity enrichment of whole-cell lysates.