iProXProteomicsXiaolong LiuRattus Rattushttp://www.iprox.org/page/project.html?id=IPX0010222000xiaolong.liu@gmail.comThe Liver Center of Fujian ProvinceiProX<h4>Background & aims</h4>Tumor neoantigens, especially cryptic antigens from non-canonical translation, are vital for cancer immunotherapy. Mass spectrometry (MS)-based de novo sequencing identifies candidates, but unverified immunogenicity and antitumor efficacy limit clinical applicability. This study aimed to identify novel non-canonical neoantigens in hepatocellular carcinoma (HCC) using MS-based de novo sequencing and rigorously validate their immunogenicity and antitumor efficacy.<h4>Methods</h4>Using a C57BL/6 subcutaneous HCC mouse model, immunopeptides were comprehensively profiled via MHC-I immunoprecipitation combined with MS-based de novo sequencing. Identified high-immunogenicity peptides predicted by deep learning were validated using ex vivo ELISpot assays. Endogenous peptide expression was confirmed using parallel reaction monitoring-targeted quantification. The antitumor efficacy of therapeutic peptide vaccines comprising the seven most immunogenic peptides combined with the adjuvant poly(I:C) was evaluated in vivo in the subcutaneous and orthotopic HCC models.<h4>Results</h4>We identified 5,576 immunopeptides, with sequence motifs consistent with prior reports. Remarkably, 95% of deep learning-predicted high-immunogenicity peptides were successfully validated by ELISpot (p <0.05). Parallel reaction monitoring confirmed endogenous expression of these peptides. Most significantly, the peptide vaccines (7 peptides + poly(I:C)) demonstrated potent antitumor efficacy in vivo compared to controls (p <0.05).<h4>Conclusions</h4>MS-based de novo sequencing combined with computational prioritization enables identification of non-canonical, immunogenic neoantigens in HCC. Selected peptides demonstrated endogenous presentation and measurable antitumor activity in preclinical models.<h4>Impact and implications</h4>This study provides robust experimental validation that mass spectrometry-based de novo sequencing effectively identifies novel, highly immunogenic non-canonical neoantigens in hepatocellular carcinoma, overcoming a key limitation of prior predictive methods and opening avenues for exploring this understudied neoantigen class in other cancers. The findings are critical for cancer immunologists and oncologists developing next-generation immunotherapies, demonstrating a viable discovery-to-validation pipeline for novel therapeutic targets. The validated neoantigens and successful peptide vaccine strategy offer a direct pathway towards developing personalized hepatocellular carcinoma immunotherapies, enabling clinicians to adopt similar integrated approaches for patient-specific neoantigen discovery; however, clinical translation beyond this preclinical murine model requires confirmation in human settings due to potential differences in HLA presentation and the tumor microenvironment.Mass spectrometry-based de novo sequencing reveals non-canonical neoantigens with antitumor efficacy in hepatocellular carcinoma.Xing Xiaohua X, Liu Mingxin M, Ouyang Jiahe J, Tang Yaxin Y, Shan Baozhen B, Tang Ruijing R, Hu En E, Li Ming M, Liu Xiaolong XfalseNeoantigen Discovery and Validation in Hepatocellular Carcinoma: Unveiling Immunogenic Targets Through Mass SpectrometryThe accurate identification of tumor neoantigens, particularly cryptic antigens from non-canonical translation, is crucial for developing effective cancer immunotherapies. However, the significance and action of cryptic antigens in anti-tumour immunity remain unclear. In this study, we utilized a highly sensitive mass spectrometry-based de novo sequencing approach to identify non-canonical MHC-binding immunopeptides and evaluate the anti-tumor immune effects of vaccines composed of these peptides in Hepatocellular carcinoma (HCC).Fri Jul 25 00:00:00 BST 2025PXD0793541011741861674