<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules"^^ . "Persistent infections with viruses such as cytomegalovirus (CMV), human immunodeficiency virus (HIV) or human papillomavirus (HPV) can lead to serious illnesses or cancer development. There are no effective therapies available to permanently eliminate these infections and cure caused diseases. With advances in understanding viral biology and biology of immune responses, one could design therapies by which the immune system is manipulated in order to eradicate the virus or the virus-induced illness. To do so, one potential approach is direct identification of viral antigen-derived epitopes, which are presented on the surface of infected or diseased cells for immune recognition. These epitopes are, in most cases, of low abundance and therefore difficult to identify. \r\nThis work presents the development of a targeted highly specific liquid chromatography-mass spectrometry (LC-MS) methodology for detection of low abundant viral epitopes from the surface of infected cells. It also offers a solution for detection of epitopes from other complicated experimental set-ups, such as identification of low abundant tumor mutation-derived epitopes. \r\nThe methodology was developed first for the detection of human leukocyte antigen (HLA)-A2-restricted HPV16 E6 and E7 epitopes, and then applied to identify HIV-derived epitopes, and mouse (m)CMV-derived epitopes presented by the mouse major histocompatibility (MHC) I complex H-2Db. The work describes the optimization of isolation, purification and enrichment of T cell epitopes for MS detection. First, HLA I-epitope complexes were immunopurified and treated with acid for dissociation of complexes. Next, epitope-containing eluates were subjected to various enrichment, purification and fractionation strategies, including ultrafiltration, normal and reverse phase chromatography, and a newly established chemical tagging strategy for epitope isolation by TiO2 pull down. Finally, epitopes were analyzed with a targeted highly specific and sensitive nano-LC-MS3 approach, where every measured peptide was manually optimized to generate the best possible spectrum. \r\nThe HPV16 E711-19 YMLDLQPET peptide was reported to be presented on HPV16-postive cell lines and tumor samples before. We were not able to identify it on the surface of HPV16-transformed cells. However, the H-2Db-restricted mCMV epitope was successfully detected in high abundance on the surface of only 1x107 cells, which is the lowest cell number ever reported for an experiment like this. The cell number could even be further reduced. Moreover, three low abundant HLA-A2-restricted HIV-derived epitopes were successfully detected on the surface of HIV-transfected cells. One of them is the first directly identified Nef-derived epitope ever reported.\r\nIn conclusion, this work demonstrates that the developed strategy for direct identification of virus-derived epitopes on the cell surface is broadly applicable to various MHC I types and virus-infected target cells. The methodology can be extended to direct identification of low abundant tumor mutation-derived epitopes. In general, directly identified epitopes form a solid base of future immunotherapy design."^^ . "2017" . . . . . . . "Renata"^^ . "Blatnik"^^ . "Renata Blatnik"^^ . . . . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (Other)"^^ . . . . . . "small.jpg"^^ . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (PDF)"^^ . . . "Renata Blatnik PhD thesis.pdf"^^ . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Development of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules (Other)"^^ . . . . . . "lightbox.jpg"^^ . . "HTML Summary of #19834 \n\nDevelopment of mass spectrometry methodology for direct identification of viral epitopes from MHC I molecules\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .