Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions |
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| Affiliation: | 1. ZIK Plasmatis, Leibniz Institute for Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-Straße 2, 17489 Greifswald, Germany;2. Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany;3. Leibniz Institute for Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-Straße 2, 17489 Greifswald, Germany;4. Greifswald University Medicine, Fleischmannstraße 8, 17475 Greifswald, Germany;5. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany;1. Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str.1, D-30625 Hannover, Germany;2. Department of Chemistry, University of Georgia, 140 Cedar Street, Athens, GA 30602-2556, USA;3. Department of Pharmacology and Toxicology, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA;4. Institute of Pharmacy, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany;5. Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str.1, D-30625 Hannover, Germany;6. Institute of Bioanalytical Chemistry, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany;1. Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium;2. Center for Biotechnology and Biomedicine, Institute for Bioanalytical Chemistry, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany;3. Institute of Statistical Science, Academia Sinica, Academia Road 128, 11529 Taipei, Taiwan;1. Université Clermont Auvergne, INRAE, UNH, Clermont-Ferrand, France;2. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany;3. Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany;3. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, 04103 Leipzig, Germany;4. Center for Biotechnology and Biomedicine, University of Leipzig, 04103 Leipzig, Germany;5. Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, 04107 Leipzig, Germany;6. Institute of Marine Research, Spanish Council for Scientific Research (IIM-CSIC), 36208 Vigo, Spain;1. School of Engineering, Sun Yat-sen University, Guangzhou 510006, China;2. Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instruments, Sun Yat-sen University, Guangzhou 510006, China;3. Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;4. Orthopaedic Institute, Soochow University, Suzhou 215006, China;5. Department of Orthopaedics, First Affiliated Hospital of Soochow University, Suzhou 215006, China |
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| Abstract: | A number of oxidative protein modifications have been well characterized during the past decade. Presumably, reversible oxidative posttranslational modifications (PTMs) play a significant role in redox signaling pathways, whereas irreversible modifications including reactive protein carbonyl groups are harmful, as their levels are typically increased during aging and in certain diseases. Despite compelling evidence linking protein carbonylation to numerous disorders, the underlying molecular mechanisms at the proteome remain to be identified. Recent advancements in analysis of PTMs by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites, but only for a limited number of proteins. Here we report the first proteome-wide study of carbonylated proteins including modification sites in HeLa cells for mild oxidative stress conditions. The analysis relied on our recent strategy utilizing mass spectrometry-based enrichment of carbonylated peptides after DNPH derivatization. Thus a total of 210 carbonylated proteins containing 643 carbonylation sites were consistently identified in three replicates. Most carbonylation sites (284, 44.2%) resulted from oxidation of lysine residues (aminoadipic semialdehyde). Additionally, 121 arginine (18.8%), 121 threonine (18.8%), and 117 proline residues (18.2%) were oxidized to reactive carbonyls. The sequence motifs were significantly enriched for lysine and arginine residues near carbonylation sites (±10 residues). Gene Ontology analysis revealed that 80% of the carbonylated proteins originated from organelles, 50% enrichment of which was demonstrated for the nucleus. Moreover, functional interactions between carbonylated proteins of kinetochore/spindle machinery and centrosome organization were significantly enriched. One-third of the 210 carbonylated proteins identified here are regulated during apoptosis. |
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| Keywords: | DNPH derivatization HILIC Laser desorption ionization (LDI) LCxLC nUPLC-Orbitrap Protein carbonylation Reactive oxygen species (ROS) |
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