Fig. 2. Detection of high confident Listeria immunopeptides.

a Filtering of Listeria peptides for detection in at least two infected samples, higher overall abundance in infected samples and absence in human proteins resulted in 68 high confident bacterial peptides from 42 Listeria protein antigens (Supplementary Data 2). b Peptide binding affinity prediction using the NetMHCpan EL 4.1 algorithm demonstrated that the large majority of the high confidence Listeria peptides are indeed predicted to bind to at least one HLA class I allele of the respective cell line^67,134–136. c A selection of 24 Listeria immunopeptide sequences was synthesized to compare their synthetic and experimental fragmentation spectra, confirming the bona fide identification of the four peptides shown here and in Supplementary Fig. 3. The correlation coefficient r is shown for each Listeria-synthetic peptide pair (see methods). d Volcano plots show that the high confident Listeria immunopeptides belong to the most upregulated immunopeptides upon infection in both cellular models. e Likewise, heat maps visualizing individual Listeria peptide intensities (z-scored) following label-free quantitation show that peptides are generally absent in the uninfected control samples, while TMT-labeling data demonstrates more low-level background intensities presumably due to ratio suppression^68,69. Immunopeptides identified by both label-free and TMT workflows are indicated by an asterisk (*), including the previously described VAYGRQVYL epitope from LLO¹³⁷. Two versions of each heat map are shown, with the left one indicating z-scored intensities after imputing empty values with low values around the detection limit (to allow t-testing). The heat maps on the right are identical, but show originally missing (unimputed) values in gray. f Venn diagrams showing the overlap between both cell lines in presented Listeria peptides (top) and their parent protein antigens (bottom). Only a single peptide (HLVPEFTNEV) was detected on both cell lines, while the overlap at the antigen level is substantially higher with seven Listeria proteins being presented by the two cellular models. Source data are provided as a Source Data file.