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-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s).

Fig. 1.

Workflow. We recently determined the peptide repertoires of the eight most frequent HLA-B27 allotypes (HLA-B*27:02 - HLA-B*27:09) by analyzing 44 independent DDA acquisitions on a TripleTOF 5600⁺ (SCIEX) mass spectrometer (18). Sequence information was obtained using the Paragon algorithm implemented in the ProteinPilot 4.5 software suite (SCIEX) and all ProteinPilot search result (.group) files were used to generate a spectral library in Skyline (MacLean et al. 2010). A custom-made retention time predictor (iRT-B27 retention time predictor) consisting of 15 intrinsic HLA-B27 and HLA-Cw4 peptides was created and used for retention time alignment (Supplemental Fig. 1). Residual, HLA-B27 allotype specific samples were pooled and analyzed by DIA/SWATH-MS on a TripleTOF 5600⁺ (SCIEX) mass spectrometer. Data were imported into Skyline and the performance of the peak picking was manually assessed for each peptide. Peptides, whose assignments failed our stringent evaluation criteria, were reanalyzed and reevaluated by MRM-MS. The quantitative data obtained by either SWATH-MS or MRM-MS were normalized for subsequent data analysis.

Fig. 2.

Cluster analysis. The 1646 quantified peptides were grouped into 30 distinct clusters according to their relative expression levels across the eight HLA-B27 allotypes. The diagrams show the relative expression level of each individual peptide within a cluster (thin gray lines). The thick red lines indicate the average expression level of all peptides within a cluster and the number of peptides within a cluster is shown below the cluster number.