Affiliations | From the ‡Ludwig Institute for Cancer Research, University of Lausanne, 1066 Epalinges, Switzerland; §Department of Oncology, University Hospital of Lausanne, 1011 Lausanne, Switzerland; **Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; ¶Service of Neurosurgery, University Hospital of Lausanne, 1011 Lausanne, Switzerland; ‖Vital IT, Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; Michal.bassani@chuv.ch. | |
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 ( = 7) and cell lines ( = 21, 10<sup>8</sup> 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 10<sup>7</sup> 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 (IFN). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFN induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications. | |