Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome
| Autor: | Sturm, Theo, Sautter, Benedikt, Wörner, Tobias P, Stevanović, Stefan, Rammensee, Hans-Georg, Planz, Oliver, Heck, Albert J R, Aebersold, Ruedi, Afd Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Biomolecular Mass Spectrometry and Proteomics |
|---|---|
| Přispěvatelé: | Afd Biomol.Mass Spect. and Proteomics, Sub Biomol.Mass Spectrometry & Proteom., Biomolecular Mass Spectrometry and Proteomics |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Lysis Chemistry(all) chemical and pharmacologic phenomena Human leukocyte antigen Mass spectrometry Major histocompatibility complex Biochemistry Chromatography Affinity Mass Spectrometry Article 03 medical and health sciences Affinity chromatography MHC class I Cysteine chemistry.chemical_classification 030102 biochemistry & molecular biology biology MHC immunoaffinity chromatography Histocompatibility Antigens Class I General Chemistry MHC bound peptides HLA ligandome mild acid elution cysteinylation post-translational modification 3. Good health 030104 developmental biology Enzyme chemistry biology.protein Peptides Protein Binding |
| Zdroj: | Journal of Proteome Research Journal of Proteome Research, 20 (1) Journal of Proteome Research, 20(1), 289. American Chemical Society |
| ISSN: | 1535-3907 1535-3893 |
| DOI: | 10.1021/acs.jproteome.0c00386 |
| Popis: | 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. ISSN:1535-3893 ISSN:1535-3907 |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|