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Validation and quantification of peptide antigens presented on MHCs using SureQuant

Nature Protocols volume 20, pages 1196–1222 (2025)Cite this article

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Abstract

Vaccines and immunotherapies that target peptide–major histocompatibility complexes (peptide–MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing vaccines and immunotherapies to target peptide–MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as ‘SureQuant’) can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition. SureQuant MHC has three main use cases: (i) conclusive confirmation of the identities of putative MHC peptides via comparison with an internal synthetic stable isotope labeled (SIL) peptide standard; (ii) accurate relative quantification by using pre-formed heavy isotope-labeled peptide–MHC complexes (hipMHCs) containing SIL peptides as internal controls for technical variation; and (iii) absolute quantification of each target peptide by using different amounts of hipMHCs loaded with synthetic peptides containing one, two or three SIL amino acids to provide an internal standard curve. Absolute quantification can help determine whether the abundance of a peptide–MHC is sufficient for certain therapeutic modalities. SureQuant MHC therefore provides unique advantages for immunologists seeking to confidently validate antigenic targets and understand the dynamics of the MHC repertoire. After synthetic standards are ordered (3–4 weeks), this protocol can be carried out in 3–4 days and is suitable for individuals with mass spectrometry experience who are comfortable with customizing instrument methods.

key points

  • Target peptides presented on MHCs can be detected by using data-dependent mass spectrometry methods, but their presence needs to be validated and quantified to develop vaccines and immunotherapies to target peptide–MHCs.

  • SureQuant is a class of mass spectrometry methods in which parallel reaction monitoring is triggered by the detection of known stable isotope-labeled standards. Standards are prepared on the basis of the DDA findings and used in this MHC SureQuant protocol.

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Fig. 1: SureQuant method overview.
Fig. 2: SureQuant MHC applications overview.
Fig. 3: SureQuant instrument method layout.
Fig. 4: SureQuant MHC can be used to validate pathogen-derived MHC peptides.
Fig. 5: SureQuant enables quantification of MHC peptides with low sample input.
Fig. 6: SureQuant enables absolute quantification of MHC peptides.

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Data availability

Raw mass spectrometry data used in Fig. 4 are part of a dataset deposited to the PRIDE database with the dataset identifier PXD03784328,62. Skyline reports containing quantification information used in Figs. 5 and 6 are included in our GitHub repository at https://github.com/oleddy/SureQuant_MHC/ and in Supplementary Information, Software 2.

Code availability

All code is available at https://github.com/oleddy/SureQuant_MHC/ and provided in Supplementary Information, Software 2.

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Acknowledgements

The authors thank all the staff members of the Ragon Institute, the Koch Institute and MIT for the essential work that they do to make our research possible. We thank C. Flower and T. Tamir for helpful conversations, training and technical guidance. L.S. and F.M.W. initially developed SureQuant MHC in collaboration with ThermoFisher Scientific. ThermoFisher Scientific provided synthetic SIL peptide synthesis services and assisted in experimental design during development of the method. A. Leshinsky, H. Amoroso and R. Cook synthesized and purified some SIL peptide standards. Other SIL standards were purchased from Biosynth. We modified code written by C. Flower to plot MS/MS spectra. TAP1 knockout THP-1 cells and a corresponding parental wild-type line were generously provided by the laboratory of W. Garcia-Beltran. This work is supported by funding from the MIT Center for Precision Cancer Research and NIH grants U01 CA238720, U54 CA283114, 1R35GM142900 and R01A1022553. This work was performed in part in the Ragon Institute BSL3 core facility, which is supported by the NIH-funded Harvard University Center for AIDS Research (P30 AI060354). We thank Y. Xie and J. Boucau for managing the facility.

Author information

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Bryan D. Bryson & Forest M. White

  2. Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA, USA

    Owen Leddy, Stefani Spranger & Bryan D. Bryson

  3. Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA

    Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Malte Roerden, Stefani Spranger & Forest M. White

  4. Aethon Therapeutics, New York, NY, USA

    Lauren Stopfer

  5. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Stefani Spranger

  6. Center for Precision Cancer Medicine, Cambridge, MA, USA

    Forest M. White

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Contributions

O.L., Y.C. and M.R. performed experiments. O.L. and Y.C. wrote code and analyzed data. O.L., Y.C., S.S., B.D.B. and F.M.W. conceptualized and planned experiments. O.L., Y.C., R.A. and F.M.W. conceptualized and planned the manuscript. L.S., O.L., Y.C., D.H.K. and F.M.W. contributed to development of the protocol. O.L., Y.C. and E.C. wrote the manuscript. O.L., Y.C., R.A., L.S. and F.M.W. revised and edited the manuscript.

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Correspondence to Forest M. White.

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Nature Protocols thanks Jennie Lill and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Leddy, O. et al. eLife 12, e84070 (2023): https://doi.org/10.7554/eLife.84070

Stopfer, L. E. et al. Proc. Natl. Acad. Sci. USA 118, e2111173118 (2021): https://doi.org/10.1073/pnas.2111173118

Supplementary information

Supplementary Software 1

Skyline templates for SureQuant MHC method building

Supplementary Software 2

Python code and example data for SureQuant MHC data analysis

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Leddy, O., Cui, Y., Ahn, R. et al. Validation and quantification of peptide antigens presented on MHCs using SureQuant. Nat Protoc 20, 1196–1222 (2025). https://doi.org/10.1038/s41596-024-01076-x

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