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Updated: Jul 6, 2026
Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis
Published on: October 15, 2021
Identification and profiling of HLA-A*02:01-restricted Toxoplasma gondii peptides through immunopeptidomics in
Ruiying Liang1,2, Xinru Luo2, Weixiang Xia1
1The Dalian Animal Virus Antigen Epitope Screening and Protein Engineering Drug Developing Key Laboratory, Dalian, China; Department of Bioengineering, College of Life and Health, Dalian University, Dalian, Liaoning, China.
HLA class I presentation of pathogen-derived peptides is essential for CD8+ T-cell recognition of Toxoplasma gondii. While in vitro MHC ligands have been documented, the in vivo ligandome during infection progression remains poorly characterized. Here, we employed an MS-based immunopeptidomics approach to directly profile the T. gondii immunopeptidome presented by HLA-A *02:01 in transgenic mice. By employing a hierarchical discovery funnel, our analysis identified a comprehensive repertoire of 3,744 unique T. gondii-derived peptides. Subsequent filtering for canonical 8-12mers, matching the typical binding length for HLA-A *02:01 ligands, established a high-confidence foundational ligandome of 3,433 peptides. Source protein analysis revealed that these peptides originate from diverse parasite proteins, including a substantial proportion of previously uncharacterized hypothetical proteins. Notably, specific ligands were consistently detected across both acute and chronic stages, suggesting stable MHC-I presentation throughout the infection cycle. By integrating in silico predictions with experimental validation, we prioritized 73 high-affinity candidates, five of which exhibited robust HLA-A *02:01 binding capacity in vitro and in vivo. Specifically, we identified a novel ligand derived from glycogen synthase (PGS) and determined its co-crystal structure with HLA-A *02:01, revealing favorable binding architecture. Overall, these findings expand the known HLA-A *02:01-restricted ligand landscape of T. gondii and provide a high-priority list of candidates for future functional validation of CD8+ T-cell immunogenicity.
HLA class I presentation of pathogen-derived peptides is essential for CD8+ T-cell recognition of Toxoplasma gondii. While in vitro MHC ligands have been documented, the in vivo ligandome during infection progression remains poorly characterized. Here, we employed an MS-based immunopeptidomics approach to directly profile the T. gondii immunopeptidome presented by HLA-A *02:01 in transgenic mice. By employing a hierarchical discovery funnel, our analysis identified a comprehensive repertoire of 3,744 unique T. gondii-derived peptides. Subsequent filtering for canonical 8-12mers, matching the typical binding length for HLA-A *02:01 ligands, established a high-confidence foundational ligandome of 3,433 peptides. Source protein analysis revealed that these peptides originate from diverse parasite proteins, including a substantial proportion of previously uncharacterized hypothetical proteins. Notably, specific ligands were consistently detected across both acute and chronic stages, suggesting stable MHC-I presentation throughout the infection cycle. By integrating in silico predictions with experimental validation, we prioritized 73 high-affinity candidates, five of which exhibited robust HLA-A *02:01 binding capacity in vitro and in vivo. Specifically, we identified a novel ligand derived from glycogen synthase (PGS) and determined its co-crystal structure with HLA-A *02:01, revealing favorable binding architecture. Overall, these findings expand the known HLA-A *02:01-restricted ligand landscape of T. gondii and provide a high-priority list of candidates for future functional validation of CD8+ T-cell immunogenicity.
