Immunopeptidomics: From Peptide Profiling to Precision Medicine

Immunopeptidomics: From Peptide Profiling to Precision Medicine In this infographic, we explore the differences between MHC class I and II antigen presentation, how immunopeptidome profiling identifies presented peptides and how these insights are shaping immunotherapy, vaccine development and precision medicine. Despite recent advances, immunopeptidomics still faces several technical and analytical challenges.9 • Low abundance of MHC-bound peptides MHC-bound peptides are typically present in very low quantities, are short in length and often lack predictable sequence motifs, making their identification difficult. • Highly complex peptide mixtures The diversity and complexity of peptide mixtures, and the need for advanced bioinformatics tools further complicate analysis. • Posttranslational modification identification Posttranslational modifications play key roles in immune recognition and are increasingly being explored as therapeutic targets. However, their unpredictable nature and low abundance make them challenging to identify and characterize. CURRENT CHALLENGES IN IMMUNOPEPTIDOMICS FUTURE DIRECTIONS In the future, further technological innovations will help to enhance the sensitivity and resolution of immunopeptidomics.9 Major histocompatibility complex (MHC) molecules play a key role in adaptive immunity. By displaying peptide fragments on the surfaces of cells, they help the immune system distinguish between healthy cells and potential threats. Continued interdisciplinary collaboration will be essential to address remaining barriers and unlock the full potential of immunopeptidomics in immunotherapy, vaccine design and biomarker discovery. Emerging models, such as patientderived organoids, could offer new ways to overcome sample limitations. AI-driven epitope prediction, integration with single-cell and spatial omics and personalized MHC profiling will enable more precise, patient-specific applications. MHC CLASS I VS CLASS II MHC molecules bind peptide fragments derived from degraded self or foreign proteins and present them on the cell surface for surveillance by T cells.2 Recognition of non-self peptides by T-cell receptors triggers T-cell activation, clonal expansion and elimination of the cell. There are two major classes of MHC molecules, each serving a distinct role in antigen presentation.3 IMMUNOPEPTIDOME IMMUNOPEPTIDOMICS The extraction and subsequent analysis of the presented peptides.1 The complete set of peptides presented by MHC molecules on the surface of cells.1 This process allows the immune system to detect and respond to infections, cancer and cellular abnormalities. Identifying the peptides presented by MHC molecules is essential for understanding immunogenicity and guiding the development of targeted immunotherapies and vaccines. PROFILING THE IMMUNOPEPTIDOME Immunopeptidomics uses mass spectrometry to directly identify peptides bound to MHC molecules, providing a snapshot of the antigens actively presented to T cells. There are several steps involved in an immunopeptidomic profiling workflow: Identified peptides are mapped to their source proteins. 3 4 5 Several factors should be considered when designing an immunopeptidomics study, including:4 Cell availability MHC expression levels Antibody availability Sample type (fresh vs frozen) Reference proteome availability 1 2 APPLICATIONS OF MHC PROFILING MHC profiling reveals the immune-visible proteome, helping researchers identify disease-associated antigens and monitor immune responses. This information can support the development of personalized immunotherapies, next-generation vaccines and safer biologic drugs. Neoantigen discovery Tumor-specific mutations can generate novel peptides (neoantigens) not found in healthy tissue. MHC class I profiling enables direct identification of these peptides, informing the design of personalized cancer immunotherapies that target tumor-specific epitopes.5, 6 Viral antigen detection MHC class I profiling can reveal viral peptides presented during infection, advancing antiviral vaccine development. Immunopeptidomics is being actively used to map SARS-CoV-2, HIV and influenza epitopes.7 Pathogen-derived epitope profiling MHC class II profiling identifies microbial peptides presented by antigen-presenting cells, guiding the design of vaccines for bacteria, parasites and other pathogens.7 Autoantigen mapping Autoimmune diseases are often driven by inappropriate presentation of self-peptides. MHC class II profiling helps identify these autoantigens in conditions such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis.4 Biologic immunogenicity prediction Biologic drugs can lose effectiveness in some patients after repeated administration due to anti-drug immune responses. MHC-associated peptide proteomics (MAPPs) detects potentially immunogenic sequences in protein therapeutics, allowing early modification to improve safety and efficacy.8 Cells or tissues are lysed. MHC-peptide complexes are isolated from cells/tissues by immunoaffinity purification. Peptides are gently eluted from the MHC. Peptides are analyzed by liquid chromatography– tandem mass spectrometry (LC-MS/MS). Several factors should be considered when designing an immunopeptidomics study, including:4 Cell availability MHC expression levels Antibody availability Sample type (fresh vs frozen) Reference proteome availability Immunopeptidomics: From Peptide Profiling to Precision Medicine In this infographic, we explore the differences between MHC class I and II antigen pr esentation, how immunopeptidome profiling identifies presented peptides and how these insights ar e shaping immunotherapy, vaccine development and precision medicine. Despite recent advances, immunopeptidomics still faces several t echnical and analytical challenges.9 • Low abundance of MHC-bound peptides MHC-bound peptides are typically present in very low quantities, are short in length and often lack predictable sequence motifs, making their identification difficult. • Highly complex peptide mixtures The diversity and complexity of peptide mixtures, and the need for advanced bioinformatics tools further complicate analysis. • Posttranslational modification identification Posttranslational modifications play key roles in immune recognition and are increasingly being explored as therapeutic targets. However, their unpredictable nature and low abundance make them challenging to identify and characterize. CURRENT CHALLENGES IN IMMUNOPEPTIDOMICS FUTURE DIRECTIONS In the future, further technological innovations will help to enhance the sensitivity and resolution of immunopeptidomics.9 Major histocompatibility complex (MHC) molecules play a key role in adaptive immunity. By displaying peptide fragments on the sur faces of cells, they help the immune system distinguish between healthy cells and potential threats. Continued interdisciplinary collaboration will be essential to address remaining barriers and unlock the full potential of immunopeptidomics in immunotherapy, vaccine design and biomarker discovery. Emerging models, such as patientderived organoids, could offer new ways to overcome sample limitations. AI-driven epitope prediction, integration with single-cell and spatial omics and personalized MHC profiling will enable more precise, patient-specific applications. MHC Class I MHC Class II Present intracellular antigens Present extracellular antigens Expressed on all nucleated cells Expressed mainly on antigen-presenting cells Interact with CD8+ T cells Interact with CD4+ T cells WRITTEN BY ANNA MACDONALD | DESIGNED BY ERIN LEMIEUX References 1. Shapiro IE, Bassani-Sternberg M. The impact of immunopeptidomics: From basic research to clinical implementation. Semin Immunol. 2023. doi: 10.1016/j.smim.2023.101727 2. Janeway CA, Travers P, Walport M, Shlomchik MJ. The major histocompatibility complex and its functions. In: Immunobiology: The Immune System in Health and Disease. 5th Edition. Garland Science; 2001. Accessed July 10, 2025. https://www.ncbi.nlm.nih.gov/books/NBK27156/ 3. Wu Y, Zhang N, Hashimoto K, Xia C, Dijkstra JM. Structural comparison between MHC classes I and II; In evolution, a class-II-like molecule probably came first. Front Immunol. 2021. doi: 10.3389/fimmu.2021.621153 4. Arshad S, Cameron B, Joglekar AV. Immunopeptidomics for autoimmunity: Unlocking the chamber of immune secrets. npj Syst Biol Appl. 2025. doi: 10.1038/s41540-024-00482-x 5. Feola S, Chiaro J, Cerullo V. Integrating immunopeptidome analysis for the design and development of cancer vaccines. Semin Immunol. 2023. doi: 10.1016/j.smim.2023.101750 6. Peltonen K, Feola S, Umer HM, et al. Therapeutic cancer vaccination with immunopeptidomics-discovered antigens confers protective antitumor efficacy. Cancers (Basel). 2021. doi: 10.3390/ cancers13143408 7. Leddy OK, White FM, Bryson BD. Leveraging immunopeptidomics to study and combat infectious disease. Nita-Lazar A, ed. mSystems. 2021. doi: 10.1128/msystems.00310-21 8. Jankowski W, Kidchob C, Bunce C, Cloake E, Resende R, Sauna ZE. The MHC associated peptide proteomics assay is a useful tool for the non-clinical assessment of immunogenicity. Front Immunol. 2023. doi: 10.3389/fimmu.2023.1271120 9. Flender D, Vilenne F, Adams C, Boonen K, Valkenborg D, Baggerman G. Exploring the dynamic landscape of immunopeptidomics: Unravelling posttranslational modifications and navigating bioinformatics terrain. Mass Spec Rev. 2025. doi: 10.1002/mas.21905