UMMS Affiliation

Department of Pathology; Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Amino Acids, Peptides, and Proteins | Hemic and Immune Systems | Immunopathology


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(b) 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 < 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(b) 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.


MHC protein, antigen presentation, mass spectrometry, peptide processing, thymic selection

Rights and Permissions

Copyright © 2021 Nanaware, Jurewicz, Clement, Lu, Santambrogio and Stern. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

DOI of Published Version



Nanaware PP, Jurewicz MM, Clement CC, Lu L, Santambrogio L, Stern LJ. Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-Ab in C57BL/6 Mouse Thymus. Front Immunol. 2021 Apr 29;12:658601. doi: 10.3389/fimmu.2021.658601. PMID: 33995376; PMCID: PMC8116589. Link to article on publisher's site

Journal/Book/Conference Title

Frontiers in immunology

Related Resources

Link to Article in PubMed

PubMed ID


Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.