title: Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.
abstract: Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.
authors.affiliation: The Rockefeller University, New York, NY 10065-6399, USA. lbozzacco@rockefeller.edu
authors.forename: Leonia
authors.initials: L
authors.lastname: Bozzacco
authors.forename: Haiqiang
authors.initials: H
authors.lastname: Yu
authors.forename: Henry A
authors.initials: HA
authors.lastname: Zebroski
authors.forename: Jörn
authors.initials: J
authors.lastname: Dengjel
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
authors.forename: Svetlana
authors.initials: S
authors.lastname: Mojsov
authors.forename: Ralph M
authors.initials: RM
authors.lastname: Steinman
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D061251:Primary Cell Culture; D013154:Spleen / Q000166:cytology* / Q000276:immunology; D053719:Tandem Mass Spectrometry
chemical_list: D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments