{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T17:12:50Z","timestamp":1781629970336,"version":"3.54.5"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,4,29]],"date-time":"2021-04-29T00:00:00Z","timestamp":1619654400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["AI146189, AI127869"],"award-info":[{"award-number":["AI146189, AI127869"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>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 core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-A<jats:sup>b<\/jats:sup> presented immunopeptidome of the thymus of C57BL\/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of &amp;lt;1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-A<jats:sup>b<\/jats:sup> binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.<\/jats:p>","DOI":"10.3389\/fimmu.2021.658601","type":"journal-article","created":{"date-parts":[[2021,4,29]],"date-time":"2021-04-29T06:07:13Z","timestamp":1619676433000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":18,"title":["Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-Ab in C57BL\/6 Mouse Thymus"],"prefix":"10.3389","volume":"12","author":[{"given":"Padma P.","family":"Nanaware","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mollie M.","family":"Jurewicz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristina 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