Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data

Abstract

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-

The Peptide Repertoire of HLA‐B27 may include Ligands with Lysine at P2 Anchor Position

“…This study resulted in three main outcomes, the first is a refinement of the C67S mutational effects on the HLA peptidomes of both HLA‐B*27:05 and HLA‐B*27:09 alleles, including also the analysis of peptidomes from HLA‐B*27:05 and HLA‐B*27:05‐C67S transgenic rats. Second, it expands our knowledge about the difference between HLA‐B*27:05 and HLA‐B*27:09 peptides (which differ only for the polymorphism Asp116His, located in the F pocket), such comparison was described already reported in somewhat smaller scales . The results described here are consistent with the previous conclusions while expanding the outcome.…”

“…The detection in the current study of 3228 peptides with P2‐Gln and 1330 peptides with P2‐Lys (in addition to the 15 450 peptides with the P2‐Arg) is likely due to the availability of much improved LC–MS/MS instruments, capable of detecting even rarely presented peptides. Previous HLA‐B*27 peptidome analyses tended to include only P2‐Arg or P2‐Gln peptides in the reported lists while discarding the poorly represented P2‐Lys peptides as fake ligands of these allotypes . The B*27‐C67S peptidome analyses described here provide, in addition to the significantly enlarged definition of the C67S peptidomes, also an indication of the extended binding of P2‐Gln and P2‐Lys peptides to this mutant and prove their positive definition as authentic B*27 ligands.…”

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

“…However, this common workflow fails to identify low‐abundant peptides that might still be important for immunotherapy development. Recently, data‐independent acquisition mass spectrometry has been applied for HLA‐I peptide discovery,41, 42 which, however, needs compatible instrumentation. Another possibility that was recently utilized for sensitive detection as well as quantification of HLA‐I peptides is targeted data acquisition 38, 43, 44, 45, 46, 47.…”

The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis

“…In contrast with this assumption, recent biochemical studies from Purcell's group, analysing a large peptide data set from the AS-associated and non-AS-associated B27 alleles expressed by transfected C1R cells, failed to identify qualitative changes in their peptide repertoire [18]. Rather, quantitative differences have been found which justify the interest towards a panel of 26 peptides eluted in lower abundance from the non-AS-associated alleles [19]. In addition, another study in which structure, peptide specificity, folding and stability of either AS-associated or non-AS-associated subtypes on C1R cells were correlated with the constitutive peptidomes, reached similar results.…”