Owing to the importance of the post-translational modifications (PTMs) of proteins in regulating biological processes, the dbPTM (http://dbPTM.mbc.nctu.edu.tw/) was developed as a comprehensive database of experimentally verified PTMs from several databases with annotations of potential PTMs for all UniProtKB protein entries. For this 10th anniversary of dbPTM, the updated resource provides not only a comprehensive dataset of experimentally verified PTMs, supported by the literature, but also an integrative interface for accessing all available databases and tools that are associated with PTM analysis. As well as collecting experimental PTM data from 14 public databases, this update manually curates over 12 000 modified peptides, including the emerging S-nitrosylation, S-glutathionylation and succinylation, from approximately 500 research articles, which were retrieved by text mining. As the number of available PTM prediction methods increases, this work compiles a non-homologous benchmark dataset to evaluate the predictive power of online PTM prediction tools. An increasing interest in the structural investigation of PTM substrate sites motivated the mapping of all experimental PTM peptides to protein entries of Protein Data Bank (PDB) based on database identifier and sequence identity, which enables users to examine spatially neighboring amino acids, solvent-accessible surface area and side-chain orientations for PTM substrate sites on tertiary structures. Since drug binding in PDB is annotated, this update identified over 1100 PTM sites that are associated with drug binding. The update also integrates metabolic pathways and protein–protein interactions to support the PTM network analysis for a group of proteins. Finally, the web interface is redesigned and enhanced to facilitate access to this resource.

Protein O-GlcNAcylation, involving the β-attachment of single N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine or threonine residues, is an O-linked glycosylation catalyzed by O-GlcNAc transferase (OGT). Molecular level investigation of the basis for OGT's substrate specificity should aid understanding how O-GlcNAc contributes to diverse cellular processes. Due to an increasing number of O-GlcNAcylated peptides with site-specific information identified by mass spectrometry (MS)-based proteomics, we were motivated to characterize substrate site motifs of O-GlcNAc transferases. In this investigation, a non-redundant dataset of 410 experimentally verified O-GlcNAcylation sites were manually extracted from dbOGAP, OGlycBase and UniProtKB. After detection of conserved motifs by using maximal dependence decomposition, profile hidden Markov model (profile HMM) was adopted to learn a first-layered model for each identified OGT substrate motif. Support Vector Machine (SVM) was then used to generate a second-layered model learned from the output values of profile HMMs in first layer. The two-layered predictive model was evaluated using a five-fold cross validation which yielded a sensitivity of 85.4%, a specificity of 84.1%, and an accuracy of 84.7%. Additionally, an independent testing set from PhosphoSitePlus, which was really non-homologous to the training data of predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (84.05%) and outperform other O-GlcNAcylation site prediction tools. A case study indicated that the proposed method could be a feasible means of conducting preliminary analyses of protein O-GlcNAcylation and has been implemented as a web-based system, OGTSite, which is now freely available at http://csb.cse.yzu.edu.tw/OGTSite/.

BackgroundThe conjugation of ubiquitin to a substrate protein (protein ubiquitylation), which involves a sequential process – E1 activation, E2 conjugation and E3 ligation, is crucial to the regulation of protein function and activity in eukaryotes. This ubiquitin-conjugation process typically binds the last amino acid of ubiquitin (glycine 76) to a lysine residue of a target protein. The high-throughput of mass spectrometry-based proteomics has stimulated a large-scale identification of ubiquitin-conjugated peptides. Hence, a new web resource, UbiSite, was developed to identify ubiquitin-conjugation site on lysines based on large-scale proteome dataset.ResultsGiven a total of 37,647 ubiquitin-conjugated proteins, including 128026 ubiquitylated peptides, obtained from various resources, this study carries out a large-scale investigation on ubiquitin-conjugation sites based on sequenced and structural characteristics. A TwoSampleLogo reveals that a significant depletion of histidine (H), arginine (R) and cysteine (C) residues around ubiquitylation sites may impact the conjugation of ubiquitins in closed three-dimensional environments. Based on the large-scale ubiquitylation dataset, a motif discovery tool, MDDLogo, has been adopted to characterize the potential substrate motifs for ubiquitin conjugation. Not only are single features such as amino acid composition (AAC), positional weighted matrix (PWM), position-specific scoring matrix (PSSM) and solvent-accessible surface area (SASA) considered, but also the effectiveness of incorporating MDDLogo-identified substrate motifs into a two-layered prediction model is taken into account. Evaluation by five-fold cross-validation showed that PSSM is the best feature in discriminating between ubiquitylation and non-ubiquitylation sites, based on support vector machine (SVM). Additionally, the two-layered SVM model integrating MDDLogo-identified substrate motifs could obtain a promising accuracy and the Matthews Correlation Coefficient (MCC) at 81.06 % and 0.586, respectively. Furthermore, the independent testing showed that the two-layered SVM model could outperform other prediction tools, reaching at 85.10 % sensitivity, 69.69 % specificity, 73.69 % accuracy and the 0.483 of MCC value.ConclusionThe independent testing result indicated the effectiveness of incorporating MDDLogo-identified motifs into the prediction of ubiquitylation sites. In order to provide meaningful assistance to researchers interested in large-scale ubiquitinome data, the two-layered SVM model has been implemented onto a web-based system (UbiSite), which is freely available at http://csb.cse.yzu.edu.tw/UbiSite/. Two cases given in the UbiSite provide a demonstration of effective identification of ubiquitylation sites with reference to substrate motifs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0246-z) contains supplementary material, which is available to authorized users.

Background: Glutarylation, the addition of a glutaryl group (five carbons) to a lysine residue of a protein molecule, is an important post-translational modification and plays a regulatory role in a variety of physiological and biological processes. As the number of experimentally identified glutarylated peptides increases, it becomes imperative to investigate substrate motifs to enhance the study of protein glutarylation. We carried out a bioinformatics investigation of glutarylation sites based on amino acid composition using a public database containing information on 430 non-homologous glutarylation sites. Results: The TwoSampleLogo analysis indicates that positively charged and polar amino acids surrounding glutarylated sites may be associated with the specificity in substrate site of protein glutarylation. Additionally, the chi-squared test was utilized to explore the intrinsic interdependence between two positions around glutarylation sites. Further, maximal dependence decomposition (MDD), which consists of partitioning a large-scale dataset into subgroups with statistically significant amino acid conservation, was used to capture motif signatures of glutarylation sites. We considered single features, such as amino acid composition (AAC), amino acid pair composition (AAPC), and composition of k-spaced amino acid pairs (CKSAAP), as well as the effectiveness of incorporating MDD-identified substrate motifs into an integrated prediction model. Evaluation by five-fold cross-validation showed that AAC was most effective in discriminating between glutarylation and non-glutarylation sites, according to support vector machine (SVM). Conclusions: The SVM model integrating MDD-identified substrate motifs performed well, with a sensitivity of 0.677, a specificity of 0.619, an accuracy of 0.638, and a Matthews Correlation Coefficient (MCC) value of 0.28. Using an independent testing dataset (46 glutarylated and 92 non-glutarylated sites) obtained from the literature, we demonstrated that the integrated SVM model could improve the predictive performance effectively, yielding a balanced sensitivity and specificity of 0.652 and 0.739, respectively. This integrated SVM model has been implemented as a web-based system (MDDGlutar), which is now freely available at http://csb.cse.yzu. edu.tw/MDDGlutar/.