AMS 3.0: prediction of post-translational modifications

Background  

We present here the recent update of AMS algorithm for identification of post-translational modification (PTM) sites in proteins based only on sequence information, using artificial neural network (ANN) method. The query protein sequence is dissected into overlapping short sequence segments. Ten different physicochemical features describe each amino acid; therefore nine residues long segment is represented as a point in a 90 dimensional space. The database of sequence segments with confirmed by experiments post-translational modification sites are used for training a set of ANNs.     

AutoMotif server: prediction of single residue post-translational modifications in proteins

The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/     

Surface accessibility of protein post-translational modifications

Protein post-translational modifications are crucial to the function of many proteins. In this study, we have investigated the structural environment of 8378 incidences of 44 types of post-translational modifications with 19 different approaches. We show that modified amino acids likely to be involved in protein-protein interactions, such as ester-linked phosphorylation, methylarginine, acetyllysine, sulfotyrosine, hydroxyproline, and hydroxylysine, are clearly surface associated. Other modifications, including O-GlcNAc, phosphohistidine, 4-aspartylphosphate, methyllysine, and ADP-ribosylarginine, are either not surface associated or are in a protein's core. Artifactual modifications were found to be randomly distributed throughout the protein. We discuss how the surface accessibility of post-translational modifications can be important for protein-protein interactivity.     

Mapping protein post-translational modifications with mass spectrometry

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.     

ProteoMod: A new tool to quantitate protein post-translational modifications

Post-translational modifications (PTMs) are known to regulate biological processes by controlling protein function. The effect of a PTM on protein function depends critically on the position and the number of modifications. While there are convenient methods available to qualitatively examine modifications like phosphorylation, glycosylation, acetylation and methylation, methods available for their quantitative assessment are cumbersome. We have developed a new tool that allows quantitation of the number of phosphorylation events in proteins with ease. The "ProteoMod" tool depends on shifts in the isoelectric points of proteins upon post-translational change. The extent of shift exhibited upon phosphorylation is algorithmically converted into the number of phosphorylations conferred. The validity of ProteoMod was confirmed by examining proteins with previously known number of phosphorylations. The list of proteins examined included HSP27, HSP70 and tumor suppressor p53. The approach can also be applied to estimate modifications like acetylation, methylation and sialylation in proteins. We analyzed shifts in isoelectric points due to sialylation events in N-glycoproteins. Using influenza hemagglutinin we show that shifts in isoelectric points correlate with intracellular distribution of this model membrane protein. In addition to extending the application of two dimensional gel electrophoresis to quantitate modifications, our study also highlights its potential use in cell biology.     

Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure–function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure-function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.     

Cell signaling, post-translational protein modifications and NMR spectroscopy

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.     

Histone Sequence Database: sequences, structures, post-translational modifications and genetic loci.

The Histone Sequence Database is an annotated and searchable collection of all available histone and histone fold sequences and structures. Particular emphasis has been placed on documenting conflicts between similar sequence entries from a number of source databases, conflicts that are not necessarily documented in the source databases themselves. New additions to the database include compilations of post-translational modifications for each of the core and linker histones, as well as genomic information in the form of map loci for the human histone gene complement, with the genetic loci linked to Online Mendelian Inheritance in Man (OMIM). The database is freely accessible through the World Wide Web at either http://genome.nhgri.nih.gov/histones/ or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES     

CONSORF: a consensus prediction system for prokaryotic coding sequences

CONSORF is a fully automatic high-accuracy identification system that provides consensus prokaryotic CDS information. It first predicts the CDSs supported by consensus alignments. The alignments are derived from multiple genome-to-proteome comparisons with other prokaryotes using the FASTX program. Then, it fills the empty genomic regions with the CDSs supported by consensus ab initio predictions. From those consensus results, CONSORF provides prediction reliability scores, predicted frame-shifts, alternative start sites and best pair-wise match information against other prokaryotes. These results are easily accessed from a website.     

Proteomic analysis of post-translational modifications

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.     

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications

Background

Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog (www.phosphortholog.com) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community.

Results

Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus.

Conclusions

PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1820-x) contains supplementary material, which is available to authorized users.     

表观遗传和蛋白质翻译后修饰在细菌耐药中的作用

日益严重的细菌耐药性有可能使人类重回前抗生素时代。细菌的耐药机理多样,深入研究细菌的耐药性形成机理有助于开发控制耐药细菌感染的新措施。表观遗传和蛋白质翻译后修饰在细胞代谢、信号转导、蛋白质降解、调控DNA复制、应激反应等方面都具有重要作用。近年来研究表明表观遗传和蛋白质翻译后修饰在细菌耐药中也扮演着重要的角色。本文总结了DNA甲基化、调控型RNAs等表观遗传因素和磷酸化、琥珀酰基化等蛋白质翻译后修饰因素在细菌耐药性中的调控作用,以期为抗生素靶标选择和抗生素开发设计提供新思路。     

Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications

DNA methylation is an important epigenetic mechanism that ensures correct gene expression and maintains genetic stability. DNA methyltransferase 1 （DNMT1） is the primary enzyme that maintains DNA methylation during replication. Dysregulation of DNMT1 is implicated in a variety of diseases. DNMT1 protein stability is regulated via various post-translational modifications, such as acetyl- ation and ubiquitination, but also through protein-protein interactions. These mechanisms ensure DNMT1 is properly activated during the correct time of the ceil cycle and at correct genomic loci, as well as in response to appropriate extracellular cues. Further understanding of these regula- tory mechanisms may help to design novel therapeutic approaches for human diseases.     

Detecting oxidative post-translational modifications in proteins

Summary. Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).     

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.     

Prediction of human protein function from post-translational modifications and localization features

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.     

Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.     

BPROMPT: A consensus server for membrane protein prediction

Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT.