Using a proteomic strategy we have previously identified Chromogranin A (ChgA) as the target antigen for the autoreactive T cell clone BDC-2.5. Additionally, we demonstrated that the peptide WE14, a naturally occurring cleavage product of ChgA, is weakly antigenic for the T cell clone in presence of antigen-presenting cells. This peptide contains a distinct amino acid sequence motif required for T cell recognition, but mass spectrometric analysis has not yet led to the identification of WE14, or a peptide containing the motif, in highly enriched antigen-containing fractions obtained from beta cell tumors. BDC-2.5 responds to antigen present in several distinct chromatographic fractions, indicating that there is more than one antigenic species. In this study, we have analyzed the two dominant antigen peaks from sequential chromatographic separation through further fractionation by peptide gel electrophoresis. Protein bands were excised from the gel and analyzed by mass spectrometric analysis and our data indicate that several proteins, including insulin and Chromogranin A (but not the peptide WE14), are present. Numerous C-peptide fragments of insulin, possibly generated through in vivo carboxypeptidase activity, could also be identified. For example, one C-Peptide cleavage product of mouse insulin 1 co-eluted with the first dominant antigen peak and the same cleavage product of mouse insulin 2 co-eluted with the second antigen peak. Additionally, various hybrid peptides covalently aggregated through bonds other than disulfide bridges could be detected. The existence of hybrid peptides formed between distinct protein cleavage products, such as the insulin C-peptide and WE14, could provide an explanation for immunological intolerance in predisposed subjects. Further work will be necessary to understand the chemical nature of the crosslinker molecules and the potential role of hybrid peptides in the development of type 1 diabetes in mice and humans.