HLA-B27 is a relative risk factor for ankylosing spondylitis (AS) and is present in about 10% in European populations but in 95% of AS patients. Various data suggest that the HLA-B27 molecule itself could be the strongest risk factor, but there is no explanation for this association. To define differential antigen presenting features of HLA-B27 in healthy individuals and AS patients, a question that cannot be addressed by biochemical studies on cell lines, the HLA-B27 protein was purified from peripheral blood lymphocytes of AS patients and healthy controls and pool sequencing of the bound peptides was performed. Results show that peptides are rich in proline (Pro) and the content of arginine (Arg) is much lower in comparison with sequences listed in the register of peptides eluted from cell cultures. Statistically significant differences were detected in frequencies of a subset of amino acids, predominantly at positions in the middle of the peptides. The frequency of Glu was increased and Gln was decreased in peptides from AS patients. Detailed analysis of purity of the immunoisolated HLA molecules excluded that the peptides might originate from any co-purified HLA molecules other than B27. We conclude that statistically significant increase in the Glu/Gln ratio of peptides from AS patients, consistent with increased deamidation in vivo, may account for differential antigenicity of HLA-B27 in patients. Source protein(s) of deamidated peptides remain unknown.