Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions

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• 作者：Ravi Chand Bollineni ; Ralf Hoffmann ; Maria Fedorova ; ^{maria.fedorova@bbz.uni-leipzig.de" class="auth_mail}
• 关键词：DCFDA ; 2 ; 7-dichlorofluorescin diacetate ; DMEM ; Dulbecco&rsquo ; s modification of Eagle&rsquo ; s medium ; DNPH ; 2 ; 4-dinitrophenyl hydrazine ; ESI ; electrospray ionization ; FBS ; fetal bovine serum ; GO ; Gene Ontology ; LDI ; laser-desorption/ionization ; OS ; oxidative stress ; PTMs ; posttranslational modifications ; RPC ; reversed-phase chromatography ; ROS ; reactive oxygen species ; HILIC ; hydrophilic interaction chromatography ; TFA ; trifluoroacetic acid ; TOF ; time-of-flight
• 刊名：Free Radical Biology and Medicine
• 出版年：March, 2014
• 年：2014
• 卷：68
• 期：Complete
• 页码：186-195
• 全文大小：1477 K

文摘

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 (卤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.