Dataset Information

Optimized liquid and gas phase fractionations increase HLA-peptidome coverage for primary cell and tissue samples

ABSTRACT: 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

ORGANISM(S): Homo Sapiens

SUBMITTER: Hasmik Keshishian

PROVIDER: PXD027165 | panorama | Mon Aug 30 00:00:00 BST 2021

REPOSITORIES: PanoramaPublic

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Publications

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

Klaeger Susan S Apffel Annie A Clauser Karl R KR Sarkizova Siranush S Oliveira Giacomo G Rachimi Suzanna S Le Phuong M PM Tarren Anna A Chea Vipheaviny V Abelin Jennifer G JG Braun David A DA Ott Patrick A PA Keshishian Hasmik H Hacohen Nir N Keskin Derin B DB Wu Catherine J CJ Carr Steven A SA

Molecular & cellular proteomics : MCP 20210812

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (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 offline followed by ion mob ...[more]

PMID: 34391888

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Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.

Dataset Information

Optimized liquid and gas phase fractionations increase HLA-peptidome coverage for primary cell and tissue samples

Publications

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

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