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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.

Original publication

DOI

10.1038/s41596-019-0133-y

Type

Journal article

Journal

Nature protocols

Publication Date

06/2019

Volume

14

Pages

1687 - 1707

Addresses

Department of Biochemistry and Molecular Biology, Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia. anthony.purcell@monash.edu.

Keywords

Cells, Cultured, Cell Line, Animals, Humans, Mice, Peptides, Histocompatibility Antigens, Histocompatibility Antigens Class I, Chromatography, Affinity, Chromatography, High Pressure Liquid, Major Histocompatibility Complex, Amino Acid Sequence, Tandem Mass Spectrometry, Chemical Precipitation