Identification Of Carbonylation Sites Research Articles

MDD-carb: a combinatorial model for the identification of protein carbonylation sites with substrate motifs

BackgroundCarbonylation, which takes place through oxidation of reactive oxygen species (ROS) on specific residues, is an irreversibly oxidative modification of proteins. It has been reported that the carbonylation is related to a number of metabolic or aging diseases including diabetes, chronic lung disease, Parkinson’s disease, and Alzheimer’s disease. Due to the lack of computational methods dedicated to exploring motif signatures of protein carbonylation sites, we were motivated to exploit an iterative statistical method to characterize and identify carbonylated sites with motif signatures.ResultsBy manually curating experimental data from research articles, we obtained 332, 144, 135, and 140 verified substrate sites for K (lysine), R (arginine), T (threonine), and P (proline) residues, respectively, from 241 carbonylated proteins. In order to examine the informative attributes for classifying between carbonylated and non-carbonylated sites, multifarious features including composition of twenty amino acids (AAC), composition of amino acid pairs (AAPC), position-specific scoring matrix (PSSM), and positional weighted matrix (PWM) were investigated in this study. Additionally, in an attempt to explore the motif signatures of carbonylation sites, an iterative statistical method was adopted to detect statistically significant dependencies of amino acid compositions between specific positions around substrate sites. Profile hidden Markov model (HMM) was then utilized to train a predictive model from each motif signature. Moreover, based on the method of support vector machine (SVM), we adopted it to construct an integrative model by combining the values of bit scores obtained from profile HMMs. The combinatorial model could provide an enhanced performance with evenly predictive sensitivity and specificity in the evaluation of cross-validation and independent testing.ConclusionThis study provides a new scheme for exploring potential motif signatures at substrate sites of protein carbonylation. The usefulness of the revealed motifs in the identification of carbonylated sites is demonstrated by their effective performance in cross-validation and independent testing. Finally, these substrate motifs were adopted to build an available online resource (MDD-Carb, http://csb.cse.yzu.edu.tw/MDDCarb/) and are also anticipated to facilitate the study of large-scale carbonylated proteomes.

Application of fluorous chemistry for the identification of carbonylation sites from oxidised proteins

Application of fluorous chemistry for the identification of carbonylation sites from oxidised proteins

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as a model protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid-phase enrichment with both CID- and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues.