Validation and quantification of peptide antigens presented on MHCs using SureQuant.

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

Journal: Nature protocols 2025;20(5):1196-1222

PMID: 39438697

Full Text on Nature Publishing Group View on PubMed

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.

Address: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA, USA.; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA.; Aethon Therapeutics, New York, NY, USA.; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA.; Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA, USA.; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA.; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.; Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].; Koch Institute for Integrative Cancer Medicine, Cambridge, MA, USA. [email protected].; Center for Precision Cancer Medicine, Cambridge, MA, USA. [email protected].

DOI: 10.1038/s41596-024-01076-x

Link outs

Free resources

Research Materials:

https://antibodies.cancer.gov/detail/CPTC-HLA-C-1

Subscription / membership required

Full Text Sources:

https://doi.org/10.1038/s41596-024-01076-x

MeSH Terms: Peptides, Humans, Major Histocompatibility Complex, Proteomics, Isotope Labeling