Incorporating support vector machine for identifying protein tyrosine sulfation sites

Tyrosine sulfation is a post‐translational modification of many secreted and membrane‐bound proteins. It governs protein‐protein interactions that are involved in leukocyte adhesion, hemostasis, and chemokine signaling. However, the intrinsic feature of sulfated protein remains elusive and remains to be delineated. This investigation presents SulfoSite, which is a computational method based on a support vector machine (SVM) for predicting protein sulfotyrosine sites. The approach was developed to consider structural information such as concerning the secondary structure and solvent accessibility of amino acids that surround the sulfotyrosine sites. One hundred sixty‐two experimentally verified tyrosine sulfation sites were identified using UniProtKB/SwissProt release 53.0. The results of a five‐fold cross‐validation evaluation suggest that the accessibility of the solvent around the sulfotyrosine sites contributes substantially to predictive accuracy. The SVM classifier can achieve an accuracy of 94.2% in five‐fold cross validation when sequence positional weighted matrix (PWM) is coupled with values of the accessible surface area (ASA). The proposed method significantly outperforms previous methods for accurately predicting the location of tyrosine sulfation sites. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Design and training of a neural network for predicting the solvent accessibility of proteins

A feed-forward neural network has been developed to predict the solvent accessibility/accessible surface area (ASA) of proteins using improved design and training methods. Several network issues ranging from the coding of ASA states to the problem of local minima of learning curve, have been addressed. Successful new approaches to overcome these problems are presented. Set of trained network weights for each ASA threshold is provided. It has been established that the prediction accuracy results with neural network are better than other reported results of ASA prediction, despite a high test to training data ratio.     

A sequence-based computational model for the prediction of the solvent accessible surface area for α-helix and β-barrel transmembrane residues

Predicting the solvent accessible surface area (ASA) of transmembrane (TM) residues is of great importance for experimental researchers to elucidate diverse physiological processes. TM residues fall into two major structural classes (α-helix membrane protein and β-barrel membrane protein). The reported solvent ASA prediction models were developed for these two types of TM residues respectively. However, this prevents the general use of these methods because one cannot determine which model is suitable for a given TM residue without information of its type. To conquer this limitation, we developed a new computational model that can be used for predicting the ASA of both TM α-helix and β-barrel residues. The model was developed from 78 α-helix membrane protein chains and 24 β-barrel membrane protein. Its prediction ability was evaluated by cross validation method and its prediction result on an independent test set of 20 membrane protein chains. The results show that our model performs well for both types of TM residues and outperforms other prediction model which was developed for the specific type of TM residues. The prediction results also proved that the random forest model incorporating conservation score is an effective sequence-based computational approach for predicting the solvent ASA of TM residues.     

Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry

Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches.     

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.     

Identification of protein methylation sites by coupling improved ant colony optimization algorithm and support vector machine

Protein methylation is involved in dozens of biological processes and plays an important role in adjusting protein physicochemical properties, conformation and function. However, with the rapid increase of protein sequence entering into databanks, the gap between the number of known sequence and the number of known methylation annotation is widening rapidly. Therefore, it is vitally significant to develop a computational method for quick and accurate identification of methylation sites. In this study, a novel predictor (Methy_SVMIACO) based on support vector machine (SVM) and improved ant colony optimization algorithm (IACO) is developed to identify methylation sites. The IACO is utilized to find the optimal feature subset and parameter of SVM, while SVM is employed to perform the identification of methylation sites. Comparison of the IACO with conventional ACO shows that the IACO converges quickly toward the global optimal solution and it is more useful tool for feature selection and SVM parameter optimization. The performance of Methy_SVMIACO is evaluated with a sensitivity of 85.71%, a specificity of 86.67%, an accuracy of 86.19% and a Matthew's correlation coefficient (MCC) of 0.7238 for lysine as well as a sensitivity of 89.08%, a specificity of 94.07%, an accuracy of 91.56% and a MCC of 0.8323 for arginine in 10-fold cross-validation test. It is shown through the analysis of the optimal feature subset that some upstream and downstream residues play important role in the methylation of arginine and lysine. Compared with other existing methods, the Methy_SVMIACO provides higher Acc, Sen and Spe, indicating that the current method may serve as a powerful complementary tool to other existing approaches in this area. The Methy_SVMIACO can be acquired freely on request from the authors.     

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long‐lived proteins. The metal‐catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off‐line nano‐liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time‐points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time‐course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step‐ladder mechanism. Copyright © 2009 John Wiley & Sons, Ltd.     

Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

GPCR‐CA: A cellular automaton image approach for predicting G‐protein–coupled receptor functional classes

Given an uncharacterized protein sequence, how can we identify whether it is a G‐protein–coupled receptor (GPCR) or not? If it is, which functional family class does it belong to? It is important to address these questions because GPCRs are among the most frequent targets of therapeutic drugs and the information thus obtained is very useful for “comparative and evolutionary pharmacology,” a technique often used for drug development. Here, we present a web‐server predictor called “GPCR‐CA,” where “CA” stands for “Cellular Automaton” (Wolfram, S. Nature 1984, 311, 419), meaning that the CA images have been utilized to reveal the pattern features hidden in piles of long and complicated protein sequences. Meanwhile, the gray‐level co‐occurrence matrix factors extracted from the CA images are used to represent the samples of proteins through their pseudo amino acid composition (Chou, K.C. Proteins 2001, 43, 246). GPCR‐CA is a two‐layer predictor: the first layer prediction engine is for identifying a query protein as GPCR on non‐GPCR; if it is a GPCR protein, the process will be automatically continued with the second‐layer prediction engine to further identify its type among the following six functional classes: (a) rhodopsin‐like, (b) secretin‐like, (c) metabotrophic/glutamate/pheromone; (d) fungal pheromone, (e) cAMP receptor, and (f) frizzled/smoothened family. The overall success rates by the predictor for the first and second layers are over 91% and 83%, respectively, that were obtained through rigorous jackknife cross‐validation tests on a new‐constructed stringent benchmark dataset in which none of proteins has ≥40% pairwise sequence identity to any other in a same subset. GPCR‐CA is freely accessible at http://218.65.61.89:8080/bioinfo/GPCR‐CA , by which one can get the desired two‐layer results for a query protein sequence within about 20 seconds. © 2008 Wiley Periodicals, Inc. J Comput Chem 2009     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified?a number of proteins in?vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in?a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest?a functional link between protein regulation by arginylation and methylation that affects nuclear structure in?vivo.     

Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.     

Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry

Protein citrullination originates from enzymatic deimination of polypeptide‐bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3‐butanedione and antipyrine. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision‐induced dissociation (CID) a specific, modification‐derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion‐based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody‐based enrichment of chemically modified citrulline‐containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future. Copyright © 2009 John Wiley & Sons, Ltd.     

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post‐translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well‐characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono‐ and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI‐MS). Despite minor matrix‐dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono‐ and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Assessment of solvent residues accessibility using three Sulfo-NHS-biotin reagents in parallel: application to footprint changes of a methyltransferase upon binding its substrate

NHS-biotin modification as a specific lysine probe coupled to mass spectrometry detection is increasingly used over the past years for assessing amino acid accessibility of proteins or complexes as an alternative when well-established methods are challenged. We present a strategy based on usage in parallel of three commercially available reagents (Sulfo-NHS-biotin, Sulfo-NHS-LC-biotin, and Sulfo-NHS-LC-LC-biotin) to efficiently assess the solvent accessibility of amino acids using MALDI-TOF mass spectrometry. The same qualitative pattern of reactivity was observed for these three reagents on the THUMPalpha protein at four reagent/polypeptide molar ratios (2 : 1, 6 : 1, 13 : 1, and 26 : 1). Peptide assignment of the detected ions gains in accuracy because of the triple redundancy due to specific increments of monoisotopic mass. These reagents are a good alternative to isotope labeling when using only a single MALDI-TOF mass spectrometer. We observed that hydroxyl groups of serine and tyrosine residues were also modified by these Sulfo-NHS-biotin reagents. The low amount of protein required and the method's simplicity make this procedure accessible and affordable in order to obtain topological information on proteins difficult to purify. This method was used to identify two lysine residues of the TrmG10 methyltransferase from Pyrococcus abyssi that were differentially reactive, modified in the protein but not in the tRNA-protein complex.     

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9‐methylated histone H3 tails

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin‐associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host–guest system between protein and protein. About 10‐ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono‐, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen‐bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen‐bonded interactions associated with aromatic cage in Me3K9 H3‐HP1 complex were observed to be much more stable than those in Me1K9 H3‐HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein–protein interactions through a trimethylated post‐translational modification. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Assessment of protein-incorporated arginine methylation in biological specimens by CZE UV-detection

Protein arginine methyltransferases methylate post-translationally arginine residues in proteins to synthesize monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine. Protein arginine methylation is involved in the regulation of signal transduction, RNA export, and cell proliferation. Moreover, upon proteolysis, arginines are released into the cytosol in which they exert important biological effects. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Quantification of these analytes is commonly performed by HPLC after sample cleanup and derivatization. We propose a CE method in which these steps have been avoided and the procedure for sample preparation has been simplified. After acidic hydrolysis of proteins, samples were dried, resuspended in water, and directly injected in CE. A baseline separation of analytes was reached in a 60 cm x 75 microm id uncoated silica capillary, by using a Tris-phosphate run buffer at pH 2.15. This method allows an accurate assessment of protein arginine methylation degree in different biological samples such as whole blood, plasma, red blood cells, cultured cells, and tissue. Moreover, its good sensitivity permits to evaluate the methylation of a single protein type after the opportune purification steps. A method applicability concerns both clinical laboratories, where the evaluation of blood protein from numerous samples could be rapidly performed, and research laboratories where the factors affecting the arginine protein methylation degree could be easily studied.