| Autor: |
Sturm T; Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland.; Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands.; Netherlands Proteomics Centre, 3584 CH Utrecht, The Netherlands.; Philochem AG, 8112 Otelfingen, Switzerland., Sautter B; Department of Immunology, Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany., Wörner TP; Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands.; Netherlands Proteomics Centre, 3584 CH Utrecht, The Netherlands., Stevanović S; Department of Immunology, Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany., Rammensee HG; Department of Immunology, Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany., Planz O; Department of Immunology, Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany., Heck AJR; Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands.; Netherlands Proteomics Centre, 3584 CH Utrecht, The Netherlands., Aebersold R; Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland.; Faculty of Science, University of Zurich, 8057 Zürich, Switzerland. |
| Abstrakt: |
To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells. |
