Mass Spectrometry for Lysine Methylation: Principles, Progress, and Prospects.

Lysine methylation is a regulatory post-translational modification with diverse roles across both histone and non-histone proteins. Despite its biological relevance, comprehensive characterization of lysine methylation remains analytically challenging due to its low stoichiometry, subtle mass changes, and the absence of standardized, robust enrichment strategies. Mass spectrometry (MS) has become the cornerstone of methylation analysis, supporting both targeted and proteome-wide investigations. In this review, we examine the evolution of MS-based workflows for lysine methylation, including advances in ionization and fragmentation techniques, high-resolution mass analyzers, and acquisition strategies such as data-independent acquisition (DIA) and parallel accumulation-serial fragmentation (PASEF). We evaluate bottom-up, middle-down, and top-down proteomic approaches and discuss enrichment methods ranging from immunoaffinity and chromatography to chemical derivatization. Particular attention is given to persistent challenges, including proteolytic constraints and isobaric interference, that complicate confident site-level resolution. Finally, we highlight emerging solutions and future directions aimed at improving the sensitivity, specificity, and reproducibility of lysine methylation profiling. Together, this synthesis provides a forward-looking roadmap for optimizing MS workflows in methyllysine proteomics.