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Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics


Peptide antigens bound to molecules encoded by the major histocompatibility complex (MHC) and presented on the cell surface form the targets of T lymphocytes. This critical arm of the adaptive immune system facilitates the eradication of pathogen-infected and cancerous cells, as well as the production of antibodies. Methods to identify these peptide antigens are critical to the development of new vaccines, for which the goal is the generation of effective adaptive immune responses and long-lasting immune memory. Here, we describe a robust protocol for the identification of MHC-bound peptides from cell lines and tissues, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUPLC–MS/MS) and recent improvements in methods for isolation and characterization of these peptides. The protocol starts with the immunoaffinity capture of naturally processed MHC-peptide complexes. The peptides dissociate from the class I human leukocyte antigens (HLAs) upon acid denaturation. This peptide cargo is then extracted and separated into fractions by HPLC, and the peptides in these fractions are identified using nUPLC–MS/MS. With this protocol, several thousand peptides can be identified from a wide variety of cell types, including cancerous and infected cells and those from tissues, with a turnaround time of 2–3 d.

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Fig. 1: MHC class I and II antigen-presentation pathways.
Fig. 2: Workflow for MHC-associated peptide purification, identification and validation by nUPLC–MS/MS.
Fig. 3: Generation of MHC immunoaffinity column.
Fig. 4: MHC immunoaffinity purification.
Fig. 5: Overview of anticipated results.


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We thank the following funding sources for supporting this work: Australian National Health and Medical Research Council (NHMRC) project grants APP1085018 and APP1084283, and Australian Research Council (ARC) project grant DP150104503. A.W.P. was supported by an NHMRC Principal Research Fellowship (APP 1137739).

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A.W.P., S.H.R. and N.T. wrote the manuscript and collated data and figures.

Corresponding authors

Correspondence to Anthony W. Purcell or Nicola Ternette.

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The authors declare no competing interests.

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Key reference(s) using this protocol

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Purcell, A.W., Ramarathinam, S.H. & Ternette, N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 14, 1687–1707 (2019).

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