Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof...

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens.Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating biorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to template and non-templated post-translational modifications, such as citrullination and carbamylation.Using this approach, we followed and imaged the in cell fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbmhyalted auto-antigen vinculin in rheumatoid arthritis.Proteolysis underpins many fundamental biological processes at every developmental stage: from the essential role of the protease fertilin during fertilization 1 to the initiation of cell death through the activation of caspases 2 . It is therefore unsurprising that dysregulation of proteolysis is a hallmark in a variety of pathologies: aberrant protease activity has been