The β-cell plays a central role in the pathogenesis of T1D by neoantigen formation due to the generation of post-translational modifications, such as deamidation, being potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 T1D-related proteins in inflammatory cytokine-stressed human islets of Langerhans. T-cell clones responsive against two Gln and three Asn deamidated peptides could be isolated from peripheral blood of T1D subjects. Ex vivo tetramer assays indicated higher T-cell frequencies in subjects with T1D compared to control subjects and a positive correlation between frequencies and insulin antibody levels at diagnosis and duration of disease for one Gln and two Asn deamidated peptides. These results highlight that inflammatory cytokine-exposed human islets are prone to both enzymatic and biochemical deamidation and suggest that both Gln and Asn deamidated peptides can promote the activation of CD4+ T cells in individuals with T1D. These findings add to the growing list of evidence that post-translational modifications undermine tolerance in T1D and may open the road for the development of new diagnostic and therapeutic applications for people living with T1D.