{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T07:50:16Z","timestamp":1775980216975,"version":"3.50.1"},"posted":{"date-parts":[[2023,3,12]]},"group-title":"Immunology","reference-count":37,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,3,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Comprehensive, in-depth identification of the human leukocyte antigen HLA-I and HLA-II tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is powerful technology for direct identification of HLA peptides from patient derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare, clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high throughput, sensitive, single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight mass spectrometry on the Bruker timsTOF SCP. We demonstrate &gt;2-fold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation and reduces input requirements to as few as 1e6 A375 cells for &gt; 800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen, and novel\/unannotated open reading frames. We also apply our optimized single-shot SCP acquisition methods to tumor derived samples, enabling sensitive, high throughput and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.<\/jats:p>","DOI":"10.1101\/2023.03.10.532106","type":"posted-content","created":{"date-parts":[[2023,3,13]],"date-time":"2023-03-13T00:15:16Z","timestamp":1678666516000},"source":"Crossref","is-referenced-by-count":4,"title":["Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry"],"prefix":"10.64898","author":[{"given":"Kshiti Meera","family":"Phulphagar","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6886-8936","authenticated-orcid":false,"given":"Claudia","family":"Ctortecka","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alvaro Sebastian","family":"Vaca Jacome","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan","family":"Klaeger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eva K.","family":"Verzani","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabrielle M.","family":"Hernandez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Namrata","family":"Udeshi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karl","family":"Clauser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Abelin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven A","family":"Carr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023032106550607000_2023.03.10.532106v2.1","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1038\/s41577-020-0306-5","article-title":"A guide to cancer immunotherapy: from T cell basic science to clinical practice","volume":"20","year":"2020","journal-title":"Nat. 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