Immunopeptidomics Services

What is the immunopeptidome?

The processing of an antigen within a cell is prerequisite for antigen recognition by T lymphocytes via their T cell receptors (TCRs). Processing describes the sequence of events that takes an intra- or extra-cellular protein in its native state and leads to presentation of proteolytically derived fragments on the cell surface bound to an MHC molecule. The cartoon shows 1) Antigen uptake, 2)Protein Processing, 3) Peptide transport with class I molecules and 4) Peptide presentation on the cell surface. The immunopeptidome is the term used to describe the complement of presented peptides on a cell surface. Immunopeptidomics is the study of the immunopeptidome.

Immunopeptidomics workflow

Cells are lysed and resulting preparations are subjected to immunoaffinity capture. Following multiple washes peptides are eluted from the MHC cleft with acid.

Purified peptides are analyzed by LC-MS/MS using nano-scale chromatography combined with an Orbitrap Astral mass spectrometer. FragPipe is used for peptide identification and quantitation.

Immunopeptidomic Profiling

To highlight the utility of our immunopeptidomics workflow we analyzed the colon cancer cell line HCT116, the human lung carcinoma cell line A549 and the human liver cancer cell line HEPG2. 10M and 100M cells were used as the input for each experiment. Immunoprecipitations were performed in triplicate. 25% of each enriched peptide sample was analyzed by LC-MS/MS using a 60 minute Data Dependent Acqusition (DDA) method. Data were processed in FragPipe at 1% FDR. The results are summarized below.

Peptide length distribution and total peptide yields for 100M and 10M cell input enrichments for A549, HCT116 and HEPG2 cells, green blue and gray bars, respectively.

Why so many 9mers?

MHC immunopeptidomics data contain many 9-mer peptides because MHC class I molecules have a closed-ended binding groove that optimally accommodates peptides of this length. Proteasomal cleavage and ERAP trimming further refine peptides to fit this preference, ensuring stable binding. 9-mers maximize interactions with MHC-I, leading to higher stability and efficient CD8+ T cell recognition.

Downloadable Immunopeptidome Profiling content

This report explores how MHC immunopeptidomics enables large-scale analysis of peptides presented by major histocompatibility complex (MHC) proteins, offering critical insights into immune system function. By leveraging advanced mass spectrometry techniques, we precisely identify and characterize MHC-bound peptides, supporting research in infection response, cancer immunotherapy, and autoimmune disease mechanisms. Learn more about our methodologies, case studies, and cutting-edge mass spectrometry capabilities to see how MHC immunopeptidomics can advance your research.

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Neoantigen Quantitation

Neoantigens are unique, mutated proteins that arise from genetic alterations in cancer cells. The immune system can sometimes recognize these altered proteins as they are presented by the MHC class I complex, triggering an immune response to target and destroy the cancer cells producing them. Immunotherapies, such as cancer vaccines or adoptive T-cell therapies, can be designed to specifically target and activate the immune system against these tumor-specific neoantigens, sparing healthy tissues and minimizing side effects. By understanding the range and diversity of neoantigens, researchers can improve the effectiveness and precision of cancer treatments, offering new hope for patients with various types of cancer.

Our workflow for neoantigen quantitation is described below.

Using human colon cancer cell line, HCT116, we showcase our ability to identify specific neoantigens and quantify their number of copies per cell. In this experiment 100M HCT116 cells were immunoprecipitated (IP) in triplicate. Peptides were eluted from enriched MHC and loaded on a C18 plate, reduced, and alkylated on column, followed by elution. 25% of each IP was analyzed on a nano LC/MS/MS using a Waters NanoAcquity system interfaced to an Orbitrap Fusion Lumos Tribrid mass spectrometer for a 2h gradient. All resulting data were processed using Skyline. Here we quantify the RBB7 N61D and PDP1 N379D neoantigens and provide copy number per cell for both WT and mutant.

This work is an extension of the following publication: Becker JP, Helm D, Rettel M, Stein F, Hernandez-Sanchez A, Urban K, Gebert J, Kloor M, Neu-Yilik G, von Knebel Doeberitz M, Hentze MW, Kulozik AE. NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes. iScience. 2021 Apr 1;24(4):102389.

Mass chromatograms for the endogenous and internal standard peptides are shown below as stacked plots.

Neoantigen (MUT) vs Wild (WT) quantitative data are summarized in charts below.

Frameshift-derived InDel Neoepitope Quantitation

Human leukocyte antigen (HLA) class I-presented peptides derived from frameshifted protein sequences are termed InDel neoepitopes (Becker et al). They allow patrolling CD8+ T cells to identify and target tumor cells presenting such InDel neoepitopes. Here we quantify the CKAP2 derived InDel peptide SLMEQIPHL in HCT116 cells and calculate the number of inDel presented peptides per cell.

Mass chromatograms for the endogenous and internal standard peptides are shown below as stacked product ion chromatograms.

InDel peptide data are summarized in the plot below.

Downloadable neoantigen quantitation content

This report shows how targeted mass spectrometry can be used to quantify presented peptides. Combined with stable labeled synthetic peptide standards the technique can be used to determine the number of peptides presented per cell.

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Some published examples of our recent MHC work

Khairkhah N, Owolabi H, Namvar A, Ibrahim M.M.H., Nyayapathy S, Jones R, Rumble J, Whitehead CE, Sebolt-Leopold JS, Everest-Dass A, Galban S. Identification of molecularly targeted therapy-induced immunopeptidome in diffuse midline glioma (DMG). Neoplasia. 73, 2026, 101278

Khan, S., Lum, J., Stephenson, H., Kohli, P. B., Mortenson, D., Ramakrishnan, D., Hung, M., Ding, S., Seto, E., Lu, S., Yen, R., Jin, D., Lee, B., Clancy, S., Schirle Oakdale, N., Novikov, N., Kang, D., Li, R., Pan, D., Dave, R., Lansdon, E., Fletcher, S. P., Garg, A. V., Thomsen, N., & Balsitis, S. (2025). A highly selective TCR-mimic antibody reveals unexpected mechanisms of HBV peptide-MHC recognition and previously unknown target biology. mAbs, 17(1), 2562998.

Skrzypczynska, K., Schimert, K., Stephenson, H., Mah, I. K., Mortenson, D., Boyd, K., Hardman, T., Novikov, N., Seto, E., Lu, S., Yen, R., Lee, B., Wang, M., Kang, D., Huang, Y., Yu, X., Hung, M., Ding, S., Thomsen, N., & Schirle Oakdale, N. (2025). Development of a PRAME pMHC targeted T cell engager for solid tumor therapy. mAbs, 17(1), 2563773.

Larson AC, Knoche SM, Brumfield GL, Doty KR, Gephart BD, Moore-Saufley PR, Solheim JC. Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells. Int J Mol Sci. 2024 Mar 11;25(6):3211.
Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells – PMC

Rana PS, Ignatz-Hoover JJ, Guo C, Mosley AL, Malek E, Federov Y, Adams DJ, Driscoll JJ. Immunoproteasome Activation Expands the MHC Class I Immunopeptidome, Unmasks Neoantigens, and Enhances T-cell Anti-Myeloma Activity. Mol Cancer Ther. 2024 Dec 3;23(12):1743-1760.
Immunoproteasome Activation Expands the MHC Class I Immunopeptidome, Unmasks Neoantigens, and Enhances T-cell Anti-Myeloma Activity – PubMed

Thrift WJ, Lounsbury NW, Broadwell Q, Heidersbach A, Freund E, Abdolazimi Y, Phung QT, Chen J, Capietto AH, Tong AJ, Rose CM, Blanchette C, Lill JR, Haley B, Delamarre L, Bourgon R, Liu K, Jhunjhunwala S. Towards designing improved cancer immunotherapy targets with a peptide-MHC-I presentation model, HLApollo. Nat Commun. 2024 Dec 30;15(1):10752.
Towards designing improved cancer immunotherapy targets with a peptide-MHC-I presentation model, HLApollo – PMC

Rana PS, Ignatz-Hoover JJ, Kim BG, Malek E, Federov Y, Adams D, Chan T, Driscoll JJ. HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity. Cancer Res Commun. 2024 Jun 18;4(6):1517-1532.
HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity – PubMed

Pyke RM, Mellacheruvu D, Dea S, Abbott C, Zhang SV, Phillips NA, Harris J, Bartha G, Desai S, McClory R, West J, Snyder MP, Chen R, Boyle SM. Precision Neoantigen Discovery Using Large-Scale Immunopeptidomes and Composite Modeling of MHC Peptide Presentation. Mol Cell Proteomics. 2023 Apr;22(4):100506.
Precision Neoantigen Discovery Using Large-Scale Immunopeptidomes and Composite Modeling of MHC Peptide Presentation – PubMed

Pyke RM, Mellacheruvu D, Dea S, Abbott CW, McDaniel L, Bhave DP, Zhang SV, Levy E, Bartha G, West J, Snyder MP, Chen RO, Boyle SM. A machine learning algorithm with subclonal sensitivity reveals widespread pan-cancer human leukocyte antigen loss of heterozygosity. Nat Commun. 2022 Apr 12;13(1):1925.
A machine learning algorithm with subclonal sensitivity reveals widespread pan-cancer human leukocyte antigen loss of heterozygosity – PubMed

Bear AS, Blanchard T, Cesare J, Ford MJ, Richman LP, Xu C, Baroja ML, McCuaig S, Costeas C, Gabunia K, Scholler J, Posey AD Jr, O’Hara MH, Smole A, Powell DJ Jr, Garcia BA, Vonderheide RH, Linette GP, Carreno BM. Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting. Nat Commun. 2021 Jul 16;12(1):4365.
Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting – PubMed

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