Type 1 diabetes is characterized by the autoimmune destruction of pancreatic ?-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 ?-cells, interferon-? (IFN-?)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant Kd-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by ?-cells grown under normal conditions or after exposure to IFN-?. These include sequences from a number of known autoantigens. Quantitation of IGRP206-214 revealed low-level presentation by K d ( 25 complexes/cell) on NIT-1 cells after IFN-? treatment compared with the simultaneous presentation of the endogenously processed Kd-restricted peptide Janus kinase-1355-363 ( 15,000 copies/cell). We have successfully sequenced peptides from NIT-1 ?-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide. ? 2012 by the American Diabetes Association.