Computational protocol: Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells

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Protocol publication

[…] Peptides were identified using Proteome Discoverer 1.4 (Thermo Scientific, Bremen, Germany). Raw Xcalibur data files were screened against the UniprotKB non-redundant protein 25.H_sapiens.fase database with a mass deviation of 20 ppm, a fragment mass tolerance of 0.8 Da and a false positive discovery rate of 95%. All identified FVIII-derived peptides with high and medium confidence were grouped and aligned for each donor. The NetMHCIIpan 3.1 server was used to determine the binding core sequence of overlapping FVIII peptides identified by mass spectrometry as outlined in the Online Supplementary Information. The binding core sequence with highest affinity was used as the representative peptide for each group of identified peptides irrespectively of the associated HLA allele. Differences in peptide numbers were statistically assessed with a two-sided non-parametric Mann-Whitney U-test using Graphpad Prism 7.0. The heat map was generated with Perseus 1.5.6.0. The absolute numbers of uniquely identified peptides for each protein were clustered with the following settings: distance - Euclidean, linkage - average, number of clusters - 300, maximal number of iterations - 10, number of restarts - 1. […]

Pipeline specifications

Software tools Proteome Discoverer, NetMHCIIpan, Perseus
Application Immune system analysis
Organisms Homo sapiens
Diseases Hemophilia A