Fig. 3: Mass spectrometry-based identification of naturally presented DNAJB1-PRKACA-derived HLA class I and HLA class II ligands. | Nature Communications

Fig. 3: Mass spectrometry-based identification of naturally presented DNAJB1-PRKACA-derived HLA class I and HLA class II ligands.

From: The oncogenic fusion protein DNAJB1-PRKACA can be specifically targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma

Fig. 3

a Schematic overview of Doxycycline (Dox)-inducible DNAJB1-PRKACA fusion gene expression. Hepatocellular carcinoma (HCC) cell lines transduced with DNAJB1-PRKACA Dox-inducible or control plasmid were treated with Dox followed by mass spectrometry (MS)-based immunopeptidome analysis of the HCC cell line, or of mature monocyte-derived dendritic cells (moDC) of a healthy volunteer (HV) incubated with HCC cell line lysate (created with BioRender.com). b Dox-induced DNAJB1-PRKACA expression by immunoblotting of HCC cell lines (HLE, SMMC-7721, and HepG2) carrying the DNAJB1-PRKACA Dox-inducible (+) or the control plasmid (−) with and without Dox treatment using an anti-Protein Kinase A (PKA) antibody (n = 3). The black arrow indicates the endogenous PKA band. GAPDH served as a loading control. c, f MS-identified peptides of c HCC cell lines (HLE n = 1, SMMC-7721 n = 3, and HepG2 n = 3) carrying the DNAJB1-PRKACA Dox-inducible or the control plasmid after Dox-treatment (data are presented as mean values ± SD) and f of mature moDCs of HVs incubated with HCC cell line lysate with or without expression of DNAJB1-PRKACA protein, respectively. d, g Distribution of MS-identified HLA ligands over the DNAJB1-PRKACA fusion protein sequence plotted with the frequency of amino acids (AA) per sample for, d HLA class I ligands of the HCC cell lines (n = 3) expressing DNAJB1-PRKACA and g HLA class II peptides of mature moDCs of HV3, HV4, and HV5 (n = 3) incubated with HCC cell line lysate expressing DNAJB1-PRKACA. e, h Fragment spectra (m/z on the x-axis) of the experimentally eluted peptides (e) of experimentally eluted HLA class I-presented ligands EIFDRYGEEV (PA*68/A*02, left) and IFDRYGEEV (PC*04/C*05, right) extracted from the DNAJB1-PRKACA expressing cell lines SMMC-7721 and HepG2, respectively (identification) by comparison to the respective synthetic peptide (validation, mirrored on the x-axis) with the calculated spectral correlation coefficient (R2). h Validation of the experimentally eluted HLA class II-presented peptide EVKEFLAKAKEDFLKK (PII-2) extracted from mature moDCs of a HV (identification) by comparison to the respective synthetic peptide isotope labeled on AA position two (validation, mirrored on the x-axis) with the calculated spectral correlation coefficient (R2). Identified b- and y-ions are marked in red and blue, respectively. Isotope-labeled ions are marked with asterisks. Source data are provided as a Source Data file.

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