Decoding Emerging Protein Post-Translational Modifications Through Chemical Biology

Protein post-translational modifications (PTMs) greatly expand proteome complexity by regulating protein activity, localization, stability, and intermolecular interactions. While advances in mass spectrometry have enabled the large-scale characterization of canonical PTMs, the discovery and analysis of newly identified rare modifications remain technically challenging because of their limited abundance, dynamic nature, and lack of robust enrichment methods. Recent advances in chemical biology have provided powerful complementary approaches that overcome many of these limitations by exploiting the chemical reactivity, metabolic origin, or enzymatic installation of PTMs. In this review, we summarize emerging chemical biology strategies that have accelerated the profiling and functional characterization of novel protein PTMs and the proteoforms they generate. We discuss metabolic labeling using bioorthogonal metabolite mimics, chemoselective derivatization of endogenous PTMs, cleavable enrichment strategies for site-specific PTM identification, proximity labeling approaches for discovering PTM-associated protein interactions, and methods for site-specific PTM installation through genetic code expansion and chemical ligation. We further highlight representative applications demonstrating how these technologies have enabled the discovery of previously unrecognized PTMs, improved proteome-wide mapping, identified PTM readers, writers, and erasers, and facilitated mechanistic studies in diverse biological and disease contexts. Finally, we discuss the strengths and limitations of current methodologies and outline future directions integrating chemical biology with quantitative proteomics, single-cell analyses, and artificial intelligence-driven data interpretation. Together, these complementary technologies are reshaping the study of protein PTMs and will continue to advance our understanding of proteome regulation in health and disease.