Discovery of Post-Translationally Modified Epitopes in Autoimmunity: Someone Has to Fish so Everyone Can Eat

Post-translational modifications (PTMs) chemically diversify the ~20,000 genomically encoded proteins into millions of functional variants, and this diversity has profound consequences for T cell-mediated immune responses. PTM-reactive T cells have been identified across numerous autoimmune diseases and, in several cases, implicated directly as pathogenic drivers. Mechanistically, PTMs promote autoimmunity through two intertwined routes: by altering peptide-MHC and TCR binding to favor recognition of self-epitopes, and by generating neoepitopes in peripheral and/or inflamed tissues that are absent from the thymus, allowing PTM-reactive T cells to escape central tolerance. This cascade is amplified by inflammatory cytokines, upregulated antigen-processing machinery, disease-associated HLA alleles, and, in some cases, exogenous triggers such as diet. We use three well-characterized examples-citrullination in multiple autoimmune disorders, hybrid insulin peptide (HIP) formation in type 1 diabetes, and gluten deamidation in celiac disease-to illustrate how distinct PTM biology, tissue specificity, and genetic encodability shape the antigenic landscape and dictate feasible discovery strategies. We then review four complementary approaches for identifying PTM-specific T cell responses: mass spectrometry-based proteomics and immunopeptidomics, computational MHC-binding prediction, antigen-directed methods, and high-throughput T cell receptor (TCR)-directed library screening methods. For each, we discuss underlying principles, strengths, and limitations, with particular attention to the challenge posed by non-canonical, PTM-bearing residues. By comparing citrullination, HIPs, and gluten deamidation across these methodologies, we highlight how biochemical alterations, tissue accessibility, and mouse-to-human conservation determine which discovery approach is most tractable. We conclude that while synthetic peptide-based methods remain the standard for validating known epitopes, emerging high-throughput, cell-based platforms are rapidly expanding the accessibility of the PTM antigenic landscape for both known and undiscovered autoimmune epitopes.