biostudies-literatureNanaware PPNational Institute of Allergy and Infectious DiseasesNIAID NIH HHS658601https://www.ebi.ac.uk/biostudies/studies/S-EPMC8116589biostudies-literatureUnknown12Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify coreFrontiers in immunologyDistinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-A^b in C57BL/6 Mouse Thymus.PMC8116589AI146189, AI127869R01 AI137198Jurewicz MMNanaware PPLu LSantambrogio LClement CCStern LJfalseDistinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-A^b in C57BL/6 Mouse Thymus.Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core2021-01-01T00:00:00Z20212025-04-04T12:33:52.799Z2022-02-10T10:35:52.91ZS-EPMC81165893399537610.3389/fimmu.2021.658601