Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.
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ABSTRACT:
SUBMITTER: Klaeger S
PROVIDER: S-EPMC8724927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Klaeger S, Apffel A, Clauser KR, Sarkizova S, Oliveira G, Rachimi S, Le PM, Tarren A, Chea V, Abelin JG, Braun DA, Ott PA, Keshishian H, Hacohen N, Keskin DB, Wu CJ, Carr SA. 2021. Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome and therefore these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.
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Project description:Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require billions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limiting amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 50 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens
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