Improvement of ubiquitylation site detection by Orbitrap mass spectrometry
| Autor: | Ype Elgersma, Lennart van der Wal, Jeroen Demmers, Erikjan Rijkers, Karel Bezstarosti, Edwin Mientjes, Dick H. W. Dekkers, Karen A. Sap |
|---|---|
| Přispěvatelé: | Biochemistry, Neurosciences, Cell Biology and Histology, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Proteomics Biophysics Peptide Orbitrap Mass spectrometry Biochemistry Mass Spectrometry law.invention 03 medical and health sciences 0302 clinical medicine Ubiquitin Clinical Protocols In vivo law Stable isotope labeling by amino acids in cell culture Methods Humans chemistry.chemical_classification Chromatography Binding Sites biology Glycylglycine Ubiquitination A-site 030104 developmental biology chemistry Cell culture 030220 oncology & carcinogenesis biology.protein Protein Processing Post-Translational HeLa Cells |
| Zdroj: | Journal of Proteomics, 172, 49-56. Elsevier Journal of proteomics, 172, 49-56. Elsevier |
| ISSN: | 1874-3919 1876-7737 |
| Popis: | Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-e-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance A K-e-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events. |
| Databáze: | OpenAIRE |
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