Fig. 3: MS and ribosome footprint-based evidence of non-canonical peptide generation.

A set of proteome-derived tumor-associated antigens, and noncHLAIp (lncRNAs and TEs), from melanoma 0D5P were synthesized in their heavy-labeled form and spiked back into replicates of HLAIp eluted from 0D5P cells to confirm the presence of endogenous HLAIp. The proportions of confirmed and non-confirmed HLAIp as determined by a PRM and b Ribo-Seq-targeted validation are shown for each of the antigen classes. c An example of the co-elution profiles of the transitions of heavy-labeled and endogenous noncHLAIp (from lncRNA; SYLRRHLDF) from 0D5P (left) is shown. The MS/MS fragmentation pattern further confirms the presence of the endogenous peptide (Δm = 10 Da) (right). d, e The Ribo-Seq profiles of two source genes show the frequency of Ribo-Seq reads from the ribosome’s P-site in three replicates. Library size-normalized P-sites per basepair are shown on a log2 scale on the y-axis, with P-sites inferred as a constant offset from the 5ʹ end of the footprint for each read length. The colored bars represent different reading frames. The yellow bars below the plots represent exons. For example, the noncHLAIp SYLRRHLDF in OVOS2 (blue arrow) falls within two nested, Ribo-Seq-supported ORFs (red arrows), within which most P-sites (red bars) fall in the first reading frame. Source data are provided as a Source Data file.