Extended Data Fig. 6. Proteogenomic analysis for detection of non-canonical peptides (NCPs) in the NSCLC cohort.

Proteogenomic analysis for detection of non-canonical peptides (NCPs) in the NSCLC cohort. a. Overview of the proteogenomic analysis. Six reading frame translation (6FT) database search was performed as previously described (Branca et al. 2014, Zhu et al. 2018) and search hits were filtered based on FDR<1%; SpectrumAI for automatic MS2 spectrum inspection/validation of single-substitution peptide identifications; and outlier expression pattern. Resulting 651 NCPs showed low identification overlap across cohort samples indicating sample specific expression. Thirteen percent of corresponding genetic loci were supported by more than one unique peptide. b. Examples of mirror plots from NCP synthetic peptide validation for a peptide that passed the manual inspection (left) and a peptide that failed the manual inspection (right). For each example the upper part shows the annotated MS2 spectrum of the NCP identified in the original proteogenomic analysis, and the lower part shows the MS2 spectrum of the corresponding synthetic peptide. In the right figure, missing fragment ions in the spectrum of the synthetic peptide are indicated. Mirror plots of all 104 NCPs that were evaluated by synthetic peptides can be found in Supplementary Data 1. c. Pie chart indicating the results of the NCP synthetic peptide validation. d. Bar plot showing the results of the NCP synthetic peptide validation for each of the six NSCLC Subtypes. In total, the 104 NCPs evaluated were identified in 156 samples (the same NCP can be identified in several samples). e. Distribution of NCP synthetic peptide validation results per subtype indicating no statistically significant difference between subtypes. P value was calculated using two-sided Fisher’s exact test.