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Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry.

Comprehensive knowledge of the HLA class I and class II peptides presented to T cells is crucial for designing innovative therapeutics against cancer and other diseases. So far, methodologies for recovery of HLA class I and II peptides for subsequent mass spectrometry-based analysis have been a major limitation. In this chapter we describe a detailed protocol for a high-throughput, reproducible, and sensitive immunoaffinity-purification of HLA-I and HLA-II peptides from up to 96 samples in a plate format, suitable for tissue samples and cell lines. Our methodology reduces sample handling, has a competitive peptide yield, and can be completed within 5 h. This simplified pipeline is applicable for basic and clinical applications.

The large-scale and in-depth identification of MHC class I- and II-presented peptides is indispensable for gaining insight into the fundamental rules of immune recognition as well as for developing innovative immunotherapeutic approaches against cancer and other diseases. In this chapter we briefly review the existing strategies for the isolation of MHC-restricted peptides and provide a detailed protocol for the immunoaffinity purification of MHC class I- and II-presented peptides from primary tissues or cells.

Ubiquitinated protein detection is often troublesome since in most cases this modification reduces the half-life of targeted proteins, inducing their degradation. Furthermore, ubiquitination is reversible thanks to the action of highly specific deubiquitinases present in all eukaryotic cells. MHC molecules ubiquitination has been demonstrated to be a key event in the regulation of the potent immunostimulatory properties of activated human dendritic cells.