Discovery of Signature Cyclic Immonium Ion for Lactyllysine Reveals Widespread and Functional Lactylation on Non-histone Proteins

Abstract Lactylation is a new modification discovered on histones. However, whether it can be installed on non-histone proteins remains unclear. Here we report the formation of a signature cyclic immonium ion of lactyllysine, together with the characteristically changed chromatographic behavior, enabling confident protein lactylation assignment by mass spectrometry. This identification strategy was confirmed by affinity-enriched lactylation proteome and revealed lactylation on nuclear non-histone proteins such as nucleolin. Subsequent exploitation of the approach to mining unenriched, deep proteome resources unveiled an understudied lactylation landscape. From the draft map of the Human Proteome, we identified widespread lactylation on DHRS7 among human tissues, and demonstrated site-directed mutagenesis of the lactylated site affects previously unannotated proteinaceous association. Additionally, the Meltome Atlas showed lactylation frequently occurs on glycolytic enzymes and concomitantly induces thermal stability changes on carrier enzymes. Collectively, the identified signatures of protein lactylation enable confident assignment and allow for the discovery of lactylation proteome expanding beyond histones, representing a step to further understand how lactylation governs cells.