Immunopeptidogenomics: Harnessing RNA-Seq to Illuminate the Dark Immunopeptidome

Human leukocyte antigen (HLA) molecules are cell-surface glycoproteins that present peptide antigens on the cell surface for surveillance by T lymphocytes, which contemporaneously seek signs of disease. Mass spectrometric analysis allows us to identify large numbers of these peptides (the immunopeptidome) following affinity purification of solubilized HLA-peptide complexes. However, in recent years, there has been a growing awareness of the "dark side" of the immunopeptidome: unconventional peptide epitopes, including neoepitopes, which elude detection by conventional search methods because their sequences are not present in reference protein databases (DBs). Here, we establish a bioinformatics workflow to aid identification of peptides generated by noncanonical translation of mRNA or by genome variants. The workflow incorporates both standard transcriptomics software and novel computer programs to produce cell line-specific protein DBs based on three-frame translation of the transcriptome. The final protein DB also includes sequences resulting from variants determined by variant calling on the same RNA-Seq data. We then searched our experimental data against both transcriptome-based and standard DBs using PEAKS Studio (Bioinformatics Solutions, Inc). Finally, further novel software helps to compare the various result sets arising for each sample, pinpoint putative genomic origins for unconventional sequences, and highlight potential neoepitopes. We applied the workflow to study the immunopeptidome of the acute myeloid leukemia cell line THP-1, using RNA-Seq and immunopeptidome data. We confidently identified over 14,000 peptides from three replicates of purified HLA peptides derived from THP-1 cells using the conventional UniProt human proteome. Using the transcriptome-based DB generated using our workflow, we recapitulated >85% of these and also identified 1029 unconventional peptides not explained by UniProt, including 16 sequences caused by nonsynonymous variants. Our workflow, which we term "immunopeptidogenomics," can provide DBs, which include pertinent unconventional sequences and allow neoepitope discovery, without becoming too large to search. Immunopeptidogenomics is a step toward unbiased search approaches that are needed to illuminate the dark side of the immunopeptidome.