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J Proteome Res. 2017 Apr 7;16(4):1806-1816. doi: 10.1021/acs.jproteome.6b00971. Epub 2017 Mar 21.

MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies.

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Department of Microbiology and Immunology, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada.
Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen , 72076 Tübingen, Germany.
Immatics Biotechnologies GmbH , 72076 Tübingen, Germany.
Department of Pathology, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada.
Proteomics Core Facility, Dalhousie University , Halifax, Nova Scotia B3H 4R2, Canada.
Department of Bio and Health Informatics, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
Centre for Innovative and Collaborative Health Services Research, IWK Health Centre , Halifax, Nova Scotia B3K 6R8, Canada.


Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.


CD8 T-cell epitopes; MHC ligandome; database searching; immunotherapy; mass spectrometry

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