Objective To evaluate similarity of the peptide repertoires bound to HLA–DR molecules that are differentially associated with rheumatoid arthritis (RA), and to define structural features of the shared peptides. Methods Peptide pools bound to HLA–DRB1*01:01, HLA–DRB1*04:01, and HLA–DRB1*10:01 (RA associated) and those bound to HLA–DRB1*15:01 (non–RA–associated) were purified and analyzed by liquid chromatography (LC) matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry (MS) and LC–ion‐trap MS. Peptide pools from each allotype were compared in terms of size, protein origin, composition, and affinity (both theoretical and experimental with some peptides). Finally, 1 peptide sequenced from DR1, DR4, and DR10, but not from DR15, was modeled in complex with all 4 HLA–DRB1 molecules and HLA–DRB5*01:01. Results A total of 6,309 masses and 962 unique peptide sequences were compared. DR10 shared 29 peptides with DR1, 9 with DR4, and 1 with DR15; DR1 shared 6 peptides with DR4 and 9 with DR15; and DR4 and DR15 shared 4 peptides. The direct identification of peptide ligands indicated that DR1 and DR10 were the most similar molecules regarding the peptides that they could share. The peptides common to these molecules contained a high proportion of Leu at P4 and basic residues at P8 binding core positions. Conclusion The degree of overlap between peptide repertoires associated with different HLA–DR molecules is low. The repertoires associated with DR1 and DR10 have the highest similarity among the molecules analyzed (∼10% overlap). Among the peptides shared between DR1 and DR10, a high proportion contained Leu4 and basic residues at the P8 position of the binding core.
Summary Major histocompatibility complex (MHC) genes are highly polymorphic, which makes each MHC molecule different regarding their peptide repertoire, so they can bind and present to T lymphocytes. The increasing importance of immunopeptidomics and its use in personalized medicine in different fields such as oncology or autoimmunity demand the correct analysis of the peptide repertoires bound to human leukocyte antigen type 1 (HLA‐I) and HLA‐II molecules. Purification of the peptide pool by affinity chromatography and individual peptide sequencing using mass spectrometry techniques is the standard protocol to define the binding motifs of the different MHC‐I and MHC‐II molecules. The identification of MHC‐I binding motifs is relatively simple, but it is more complicated for MHC‐II. There are some programs that identify the anchor motifs of MHC‐II molecules. However, these programs do not identify the anchor motif correctly for some HLA‐II molecules and some anchor motifs have been deduced using subjective interpretation of the data. Here, we present a new software, called PRBAM (Peptide Repertoire‐Based Anchor Motif) that uses a new algorithm based on the peptide–MHC interactions and, using peptide lists obtained by mass spectrometry sequencing, identifies the binding motif of MHC‐I and HLA‐DR molecules. PRBAM has an easy‐to‐use interface, and the results are presented in graphics, tables and peptide lists. Finally, the fact that PRBAM uses a new algorithm makes it complementary to other existing programs.
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