Abstract
Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n = 7) and cell lines (n = 21, 108 cells per replicate), which resulted in unprecedented depth, sensitivity and high reproducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method's achieved sensitivity, even single measurements of peptides purified from 107 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFNγ). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFNγ induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications.
Highlights
From the ‡Ludwig Institute for Cancer Research, University of Lausanne, 1066 Epalinges, Switzerland; §Department of Oncology, University Hospital of Lausanne, 1011 Lausanne, Switzerland; ¶Service of Neurosurgery, University Hospital of Lausanne, 1011 Lausanne, Switzerland; ʈVital IT, Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; **Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland
Gibbs Clustering Analysis for human leukocyte antigen (HLA)-II Peptides—Gibbscluster-2.0e [31] was run independently for each sample using all HLA-IIp identified in a given sample, with the default options except that the number of clusters was tested between 1 and 6, the number of seeds for initial conditions was set to 5, the initial Monte Carlo temperature was 1.5, and we enabled the preference for hydrophobic a.a. at P1
Immunopeptidomics and Proteomics Capture Similar Global Changes on IFN␥ Treatment—We further explored qualitative global changes in the HLA-bound peptides (HLAp) repertoire modulated on IFN␥ treatment and detected 1157 HLA-Ip that were significantly up-regulated and 551 down-regulated HLA-Ip (t test false discovery rate (FDR) ϭ 0.01, S0 ϭ 1)
Summary
In this work we set out to develop the first high-throughput method for IP of HLAp for MS-based immunopeptidomics, suitable for both basic and translational studies, where thousands of unique HLA-Ip and -IIp can be readily identified in a single IP procedure from cell lines and tissue samples. To exclude carry-over between wells during the affinity purification steps, we incorporated in plate 1 cross-linked beads not loaded with lysate (mock samples), and no HLA-I or HLA-II complexes were detected here (supplemental Fig. S1A).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have