MAPRes: an efficient method to analyze protein sequence around post-translational modification sites

Functional switches are often regulated by dynamic protein modifications. Assessing protein functions, in vivo, and their functional switches remains still a great challenge in this age of development. An alternative methodology based on in silico procedures may facilitate assessing the multifunctionality of proteins and, in addition, allow predicting functions of those proteins that exhibit their functionality through transitory modifications. Extensive research is ongoing to predict the sequence of protein modification sites and analyze their dynamic nature. This study reports the analysis performed on phosphorylation, Phospho.ELM (version 3.0) and glycosylation, OGlycBase (version 6.0) data for mining association patterns utilizing a newly developed algorithm, MAPRes. This method, MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications), is based on mining association among significantly preferred amino acids of neighboring sequence environment and modification sites themselves. Association patterns arrived at by association pattern/rule mining were in significant conformity with the results of different approaches. However, attempts to analyze substrate sequence environment of phosphorylation sites catalyzed for Tyr kinases and the sequence data for O-GlcNAc modification were not successful, due to the limited data available. Using the MAPRes algorithm for developing an association among PTM site with its vicinal amino acids is a valid method with many potential uses: this is indeed the first method ever to apply the association pattern mining technique to protein post-translational modification data.     

Influence of the sequence environment and properties of neighboring amino acids on amino‐acetylation: Relevance for structure‐function analysis

Proteins function is regulated by co‐translational modifications and post‐translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post‐translationally on the ε‐amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co‐translationally on Ser, Thr, Gly, Met, and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for PTMs). MAPRes was utilized to examine the sequence patterns vicinal to modified and non‐modified residues, taking into account their charge and polarity. The PTMs data were further sub‐divided according to their sub‐cellular location (nuclear, mitochondrial, and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non‐acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure‐function relationship. J. Cell. Biochem. 114: 874–887, 2013. © 2012 Wiley Periodicals, Inc.     

Phosphoproteome sequence analysis and significance: Mining association patterns around phosphorylation sites utilizing MAPRes

Phosphorylation, one of the most common protein post‐translational modifications (PTMs) on hydroxyl groups of S/T/Y is catalyzed by kinases and involves the presence or absence of certain amino acid residues in the vicinity of the phosphorylation sites. Using MAPRes, we have analyzed the substrate proteins of Phospho.ELM 7.0 and found that there are both general and specific requirements for the presence or absence of particular amino acids in the vicinity of phosphorylated S/T/Y for both of the phosphorylation data, whether or not kinase information was taken into account. Patterns extracted by MAPRes for kinase‐specific data have been utilized to find the consensus sequence motifs for various kinases required to catalyze the process of phosphorylation on S/T/Y. These consensus sequences for different kinase groups, families, and individual members are consistent with those described earlier with some novel consensus reported for the first time. A comparison study for the patterns mined by MAPRes with the results of existing prediction methods was performed by searching for these patterns in the vicinity of phosphorylation sites predicted by different available method. This comparison resulted in 87–98% conformity with the results of the predictions by available methods. Additionally, the patterns mined by MAPRes for substrate sites included 61 kinases, the highest number analyzed so far. J. Cell. Biochem. 108: 64–74, 2009. © 2009 Wiley‐Liss, Inc.     

Cover Picture: Proteomics 10/2008

In this issue of Proteomics you will find the following highlighted articles: Open‐pit mining for compatible neighbors Open pit mines are an explosive topic in some parts of the US and elsewhere around the world. In this case, however, it is information, not coal or copper, that is being mined from computer files. Ahmad et al. are looking for patterns in the sequence of amino acids that surround a landmark, an amino acid that is frequently modified by phosphorylation or glycosylation. If an appropriate set of rules can be found, it becomes feasible to predict sites of post‐translational modification (PTM) and possibly winners in conflicts from overlapping sites. Algorithms (MAPRes) for O‐glycosylation (GalNAc) and O‐phosphorylation have been implemented that show good fit, correlating well with patterns predicted by existing software. The MAPRes software should also be useful in creating patterns for features such as protease targets and secondary protein structures. Ahmad, I. et al., Proteomics 2008, 8, 1954–1958. Synthetic sequence steals enzyme‐specific (PKCα) spot It is interesting that evolution has optimized, rather than maximized, many interactions. It was only after we maximized these interactions artificially that we began to recognize the subtleties possible with control systems that were not pushed to the max full time. On the other hand, less than 100% is not satisfactory if we are trying to clean out metastasizing tumor cells. Kang et al. are looking for maximum discrimination between protein kinase C (PKC) isozymes for diagnostic and therapeutic applications. PKCα is normally involved in differentiation, growth, and programmed death of many cell types. The researchers began by designing and screening a set of >1700 PKCα target peptides. They selected the one with the highest efficiency of being labeled and characterized it further for kinetics (K_m, and V_max) with 11 PKC isozymes. They also used it for Western blot evaluation of enzyme levels in tumor and normal tissues. Kang, J.‐H. et al., Proteomics 2008, 8, 2006–2011. Subtleties of B. subtilis biological labeling Bacilllus subtilis is a workhorse bacterium, if you'll allow a mixed metaphor. My grad school friends who worked with it always claimed it was a “higher organism” than E. coli because it could differentiate, sort of like yeast. Because it is well studied genetically and physiologically, it has been adopted as a useful model system for the study of stress responses. Dreisbach et al. wanted to extend proteome analysis to membrane proteins under different starvation conditions that generated the stringent response. Conventional methods (e.g. 2‐DE) were not quantitative enough, or had unacceptable error rates (in vitro labelling). They found in vivo labelling with either specific amino acids (SILAC with lysine) or general metabolic labelling (14N/15N‐metabolic) to meet their needs. Samples could be mixed with controls prior to extraction and digestion to markedly reduce technical error rates. Both methods were considered suitable for quantitative proteomic analysis of membrane proteins. Dreisbach, A. et al., Proteomics 2008, 8, 2062–2076.     

Current strategies and findings in clinically relevant post-translational modification-specific proteomics

Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research.     

In this issue: Proteomics 10/2008

In this issue of Proteomics you will find the following highlighted articles: Open‐pit mining for compatible neighbors Open pit mines are an explosive topic in some parts of the US and elsewhere around the world. In this case, however, it is information, not coal or copper, that is being mined from computer files. Ahmad et al. are looking for patterns in the sequence of amino acids that surround a landmark, an amino acid that is frequently modified by phosphorylation or glycosylation. If an appropriate set of rules can be found, it becomes feasible to predict sites of post‐translational modification (PTM) and possibly winners in conflicts from overlapping sites. Algorithms (MAPRes) for O‐glycosylation (GalNAc) and O‐phosphorylation have been implemented that show good fit, correlating well with patterns predicted by existing software. The MAPRes software should also be useful in creating patterns for features such as protease targets and secondary protein structures. Ahmad, I. et al., Proteomics 2008, 8, 1954–1958. Synthetic sequence steals enzyme‐specific (PKCα) spot It is interesting that evolution has optimized, rather than maximized, many interactions. It was only after we maximized these interactions artificially that we began to recognize the subtleties possible with control systems that were not pushed to the max full time. On the other hand, less than 100% is not satisfactory if we are trying to clean out metastasizing tumor cells. Kang et al. are looking for maximum discrimination between protein kinase C (PKC) isozymes for diagnostic and therapeutic applications. PKCα is normally involved in differentiation, growth, and programmed death of many cell types. The researchers began by designing and screening a set of >1700 PKCα target peptides. They selected the one with the highest efficiency of being labeled and characterized it further for kinetics (K_m, and V_max) with 11 PKC isozymes. They also used it for Western blot evaluation of enzyme levels in tumor and normal tissues. Kang, J.‐H. et al., Proteomics 2008, 8, 2006–2011. Subtleties of B. subtilis biological labeling Bacilllus subtilis is a workhorse bacterium, if you'll allow a mixed metaphor. My grad school friends who worked with it always claimed it was a “higher organism” than E. coli because it could differentiate, sort of like yeast. Because it is well studied genetically and physiologically, it has been adopted as a useful model system for the study of stress responses. Dreisbach et al. wanted to extend proteome analysis to membrane proteins under different starvation conditions that generated the stringent response. Conventional methods (e.g. 2‐DE) were not quantitative enough, or had unacceptable error rates (in vitro labelling). They found in vivo labelling with either specific amino acids (SILAC with lysine) or general metabolic labelling (14N/15N‐metabolic) to meet their needs. Samples could be mixed with controls prior to extraction and digestion to markedly reduce technical error rates. Both methods were considered suitable for quantitative proteomic analysis of membrane proteins. Dreisbach, A. et al., Proteomics 2008, 8, 2062–2076.     

Dual Coordination of Post Translational Modifications in Human Protein Networks

Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling.     

Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

HISTOCHEMISTRY OF THE PRIMARY THICKENING MERISTEM IN THE VEGETATIVE STEM OF ALLIUM CEPA L.

Stems of Allium cepa L., 1, 2, 5, and 6 months old respond similarly when stained for protein and RNA. The primary thickening meristem (PTM) stains more intensely than surrounding stem tissues. The acropetal region of the PTM is a broadly staining band which narrows basipetally to the level of the initiation of shoot-borne roots in the stem and disappears more basipetally. These staining patterns are consistent with the hypothesis that the PTM functions in stem thickening and root production, and also indicate that the meristem functions before histological evidence of the cambial-like zone exists in the onion stem. Histochemical staining may be an accurate method of locating the PTM.     

Engineering Proteases for Mass Spectrometry‐Based Post Translational Modification Analyses

Protein post‐translational modifications (PTMs) are important modulators of virtually all cellular processes, and frequently correlate with not only the rate but also severity of diseases. There has been considerable interest to map all possible PTM sites to be used as drug targets. Current approaches for PTM analysis suffer from a number of challenges; one of which is the lack of a PTM specific cleaving reagent. A central technology for global quantitative PTM analysis, mass spectrometry (MS) based proteomics, is biased toward trypsin due to its high activity and specificity. This bias becomes a problem when a PTM is located at or near tryptic cleavage sites, in which case the PTM might block recognition by trypsin, resulting in missed cleavage and sequence coverage gaps. Reviewed here are recent advances in engineering new proteases for PTM analyses, and how these new proteases are beginning to address current challenges in the field.     

Global substrate specificity profiling of post‐translational modifying enzymes

Enzymes that modify the proteome, referred to as post‐translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide‐based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP‐MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems‐level understanding of PTM enzyme regulation and function.     

Identification of PTM5 protein interaction partners, a MADS-box gene involved in aspen tree vegetative development

In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees, Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonal expression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots. Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5 protein-protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like other SOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studies showed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cell growth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes with specific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.     

马铃薯块茎蛾生物学、生态学与综合治理

马铃薯块茎蛾又称烟草潜叶蛾Phthorimaea operculella,起源于中美洲和南美洲北部地区,现已分布在亚洲、欧洲、北美洲、非洲等100多个国家,是茄科作物的世界性农业害虫,尤其对马铃薯有毁灭性的危害。目前,该虫在我国南方马铃薯产区普遍发生,尤其是在云南、四川、贵州等地区该害虫发生极为严重,且随着气候的变化该虫可能会扩散到其他马铃薯生产区。马铃薯块茎蛾主要进行两性生殖,少数孤雌生殖,其幼虫钻蛀叶片和薯块危害。初孵幼虫无性二态性,4龄幼虫、蛹和成虫均可依据外形特征进行雌雄区分。马铃薯块茎蛾发生世代数取决于当地的农业气候条件,年发生2~12代。马铃薯块茎蛾对温度有广泛的适应性,且在干燥炎热的年份该虫容易大爆发。马铃薯块茎蛾早期防控主要集中在种植抗性品种、深种、灌溉等农业防治措施上,但化学防治依然是马铃薯生产过程中防治马铃薯块茎蛾的主要方式,由于化学农药的广泛使用,该虫对有机磷类、拟除虫菊酯类等杀虫剂均产生了不同程度的抗性;为了减少化学农药的使用,延缓抗药性的发展,发现并筛选到多种对马铃薯块茎蛾具有防治作用的天敌昆虫和昆虫病原微生物。以(E4,Z7) 十三碳二烯基乙酸酯和(E4,Z7,Z10) 十三碳三烯基乙酸酯为主要成分的马铃薯块茎蛾性信息素在马铃薯块茎蛾监测和防治中也取得了较好的效果。桉树、皱叶薄荷等植物源化合物能够抑制马铃薯块茎蛾产卵;转基因抗虫马铃薯、遗传不育技术等绿色防控技术也成为了防控马铃薯块茎蛾的新方法。以往的研究发现使用单一生物防治手段很难达到理想的防控效果,集成与生物防控技术相容的化学物质、自然天敌和病原微生物等技术是有效控制马铃薯块茎蛾种群的重要趋势。本文系统综述了国内外马铃薯块茎蛾发生为害规律及综合防控技术研究进展,以期为马铃薯块茎蛾的持续治理提供参考依据。     

Antibacterial sulfur-containing platensimycin and platencin congeners from Streptomyces platensis SB12029

The platensimycin (PTM) and platencin (PTN) class of natural products are promising drug leads that target bacterial and mammalian fatty acid synthases. Natural congeners and synthetic analogues of PTM and PTN have been instrumental in determining their structure–activity relationships, with only a few analogues retaining the potencies of PTM and PTN. Here we describe the identification and isolation of two new sulfur-containing PTM congeners (3 and 5) from the engineered dual PTM–PTN overproducing Streptomyces platensis SB12029. Structure elucidation of platensimycin D1 (5), a sulfur-containing PTM pseudo-dimer, revealed the existence of its presumptive thioacid precursor (3). The unstable thioacid 3 was isolated and confirmed by structural characterization of its permethylated product (6). LC–MS analysis of crude extracts of SB12029 confirmed the presence of the thioacid analogue of PTN (4). The minimum inhibitory concentration (MIC) was determined for 5 revealing retention of the strong antibacterial activity of PTM.     

SysPTM: A Systematic Resource for Proteomic Research on Post-translational Modifications

With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user''s PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.Post-translational modifications (PTMs)¹ are various processing events that change the maturity, activity, and/or turnover of proteins. More than 200 different types of PTMs have been found, with new ones still being reported (1). PTMs not only change the physicochemical properties of proteins (2) but also dynamically regulate various biological events such as protein degradation, subcellular localization, conformational change, protein-protein interaction, and signal transduction (3–5). Previous studies have revealed the central roles of PTMs in human health and disease. For example, phosphorylation of pRB1 has been associated with tumorigenesis through controlling cell division (6); S-nitrosylation of parkin regulates its E3 ligase activity, resulting in protein accumulation in sporadic Parkinson disease (7); and defects in protein glycosylation have been related to several forms of congenital muscular dystrophy (8). Given this important role in health and disease, PTMs have been regarded as potential disease biomarkers or therapeutic targets. For example, Erlotinib (Tarceva), an inhibitor of epidermal growth factor receptor tyrosine kinase, has been approved by the Food and Drug Administration to treat non-small cell lung cancer (9); and histone deacetylase inhibitors have been demonstrated to have a potential therapeutic role in Huntington disease (10). The broad range of important roles played by PTMs in physiological and pathological processes has made PTM research an active field in recent years. Yet we remain limited in our knowledge of the full scope of PTM distribution on proteins and the precise location of PTM sites.There are two major kinds of experimental methods to identify PTMs: 1) traditional biological experiments such as radiolabeling PTM proteins (11), Western analysis with antibodies against specific modifications (12), and site-directed mutagenesis of potential modification sites (13); and 2) large-scale proteomic experiments, especially multiple-dimensional liquid chromatography tandem mass spectrometry. Traditional experiments are laborious and time-consuming, resulting in slow data accumulation. By contrast, more recent MS/MS experiments have led to the discovery of thousands of new phosphorylation (14), glycosylation (15), acetylation (16), sumoylation (17), S-nitrosylation (18), and other modification sites. For example, based on MS/MS data, more than 6,000 phosphopeptides have been reported in HeLa cells (14), and 159 candidate sumoylated proteins have been found in yeast (17). Although advanced technologies have allowed PTM data to accumulate rapidly, it is impossible to identify all PTM sites for a set of proteins in one experiment, due to biased modification enrichment related to experimental protocol, limited sensitivity of mass spectrometer instrumentation, and failures in spectrum matching. Data bases are needed to amass PTM data from various experiments for comprehensive understanding of PTMs.Most data bases for storing PTM information have fallen into two general classes. One class focuses on a single modification type, such as Phospho.ELM (19) for phosphorylation or O-GLYCBASE (20) for glycosylation. Although these data bases have been widely used, they are limited in utility due to recording only a single modification type. The other class of PTM data base is the primary protein data base; these data bases collect PTM information with multiple modification types but are more broadly focused on providing diverse information about proteins, rather than PTM information specifically. Swiss-Prot (21) and HPRD (22) are examples of such data bases. As compared with either of the above two types of data base, integrated PTM data bases are more desirable. One example is dbPTM (23), which integrated experimentally determined PTM information from four external data bases. PhosphoSite started the harvesting of phosphorylation sites from published literature with a focus on in vivo mammalian phosphorylation data (24), but recently it has expanded to integrate nine other modification types. Even integrated data bases, however, have not taken into full consideration the aforementioned quickly accumulating PTM data from MS/MS experiments. These data, many of which are reported in the published literature but not collected in any data base, continue to increase rapidly due to new experiments. Such a wealth of information should be incorporated more comprehensively into the current PTM knowledge domain.At the same time, the high-throughput nature and complexity of MS/MS data pose computational challenges for proteome-scale PTM analyses in a biological context. A pure data repository is insufficient for such tasks. Powerful computational tools must accompany data repositories to allow knowledge extraction.To address these needs, we developed a systematic resource for PTM research, SysPTM, consisting of a PTM data base and four analysis tools. The SysPTM data base incorporates the existing features of numerous previous data bases, with an emphasis on collecting modification datasets from MS/MS experiments reported in the literature. The current release of SysPTM (v1.1) contains data detailing 117,349 PTM sites on 33,421 proteins involving nearly 50 modification types. The four analysis tools are PTMBlast, PTMPathway, PTMPhylog, and PTMCluster, which, respectively, can compare user PTM datasets with PTM data stored in SysPTM, map PTM proteins to KEGG pathways, discover potentially conserved PTM sites, and find significant clusters of multi-site modifications.In this work, an in-house MS/MS phosphorylation dataset from mouse embryonic stem cells was analyzed to demonstrate the SysPTM workflow. SysPTM can be accessed online.     

Deciphering a global network of functionally associated post‐translational modifications

Various post‐translational modifications (PTMs) fine‐tune the functions of almost all eukaryotic proteins, and co‐regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co‐evolution within proteins based on the co‐occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co‐evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane‐associated proteins and in the context of particular protein domains and short‐linear motifs. The global network of co‐evolving PTM types implies a complex and intertwined post‐translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.     

High-throughput profiling of posttranslational modification enzymes by phage display

Phage display has been used as a high-throughput platform for identifying proteins or peptides with desired binding or catalytic activities from a complex proteome. Recently, phage display has been applied to profile the catalytic activities of posttranslational modification (PTM) enzymes. Here, we highlight recent work elucidating the downstream targets of PTM enzymes by phage display, including the genome-wide profiling of biosynthetic enzymes subject to phosphopantetheinyl transferase (PPTase) modification.     

Progress in epigenetic histone modification analysis by mass spectrometry for clinical investigations

Chromatin biology and epigenetics are scientific fields that are rapid expanding due to their fundamental role in understanding cell development, heritable characters and progression of diseases. Histone post-translational modifications (PTMs) are major regulators of the epigenetic machinery due to their ability to modulate gene expression, DNA repair and chromosome condensation. Large-scale strategies based on mass spectrometry have been impressively improved in the last decade, so that global changes of histone PTM abundances are quantifiable with nearly routine proteomics analyses and it is now possible to determine combinatorial patterns of modifications. Presented here is an overview of the most utilized and newly developed proteomics strategies for histone PTM characterization and a number of case studies where epigenetic mechanisms have been comprehensively characterized. Moreover, a number of current epigenetic therapies are illustrated, with an emphasis on cancer.