Identification and Quantitation of MHC Class II-Bound Peptides from Mouse Spleen Dendritic Cells by Immunoprecipitation and Mass Spectrometry Analysis
Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that could be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipitation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.
Key wordsEndogenous peptides MHC DCs Flt3L LC-MS/MS MASCOT search engine Quantitation analysis Isotope-labeled peptides
We thank Dr. Svetlana Mojsov, the Rockefeller University, New York and Dr. Jörn Dengjel, University of Freiburg, Freiburg, for critical discussion and analytical tools. We are grateful to Marguerite Nulty for help with the references. This work was supported by grants from National Institute of Allergy and Infectious Diseases (NIAID), AI13013, 081677, and AI051573 to Ralph M. Steinman.
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