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

ABSTRACT 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.