Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

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.     

THANATOS: an integrative data resource of proteins and post-translational modifications in the regulation of autophagy

Macroautophagy/autophagy is a highly conserved process for degrading cytoplasmic contents, determines cell survival or death, and regulates the cellular homeostasis. Besides ATG proteins, numerous regulators together with various post-translational modifications (PTMs) are also involved in autophagy. In this work, we collected 4,237 experimentally identified proteins regulated in autophagy and cell death pathways from the literature. Then we computationally identified potential orthologs of known proteins, and developed a comprehensive database of The Autophagy, Necrosis, ApopTosis OrchestratorS (THANATOS, http://thanatos.biocuckoo.org), containing 191,543 proteins potentially associated with autophagy and cell death pathways in 164 eukaryotes. We performed an evolutionary analysis of ATG genes, and observed that ATGs required for the autophagosome formation are highly conserved across eukaryotes. Further analyses revealed that known cancer genes and drug targets were overrepresented in human autophagy proteins, which were significantly associated in a number of signaling pathways and human diseases. By reconstructing a human kinase-substrate phosphorylation network for ATG proteins, our results confirmed that phosphorylation play a critical role in regulating autophagy. In total, we mapped 65,015 known sites of 11 types of PTMs to collected proteins, and revealed that all types of PTM substrates were enriched in human autophagy. In addition, we observed multiple types of PTM regulators such as protein kinases and ubiquitin E3 ligases or adaptors were significantly associated with human autophagy, and again the results emphasized the importance of PTM regulations in autophagy. We anticipated THANATOS can be a useful resource for further studies.     

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。     

A peptidoform based proteomic strategy for studying functions of post-translational modifications

Protein post-translational modifications (PTMs) generate an enormous, but as yet undetermined, expansion of the produced proteoforms. In this Viewpoint, we firstly reviewed the concepts of proteoform and peptidoform. We show that many of the current PTM biological investigation and annotation studies largely follow a PTM site-specific rather than proteoform-specific approach. We further illustrate a potentially useful matching strategy in which a particular “modified peptidoform” is matched to the corresponding “unmodified peptidoform” as a reference for the quantitative analysis between samples and conditions. We suggest this strategy has the potential to provide more directly relevant information to learn the PTM site-specific biological functions. Accordingly, we advocate for the wider use of the nomenclature “peptidoform” in future bottom-up proteomic studies.     

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine,two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

ABSTRACT

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.     

Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins

The human salivary acidic proline-rich proteins (aPRPs) complex was investigated by different chromatographic and mass spectrometric approaches and the main aPRPs, namely PRP-1, PRP-2 and PIF-s (15,515 amu), Db-s (17,632 amu) and Pa (15,462 amu) proteins, were detected. All these isoforms are phosphorylated at Ser-8 and Ser-22 and have a pyroglutamic moiety at the N-terminus. Apart from Pa, all the other aPRPs undergo a proteolytic cleavage at Arg-106 residue (Arg-127 in Db-s protein), that generates the small PC peptide (4371 amu) and PRP-3, PRP-4, PIF-f (11,162 amu) and Db-f (13,280 amu) proteins, all of which were detected. With regard to the Pa protein, the main form detected was the dimeric derivative (Pa 2-mer, 30,922 amu) originated by a disulfide bond involving Cys-103 residue. Besides these known isoforms, several previously undetected aPRP derivatives were found (in minor amounts): (i) the triphosphorylated derivatives of PRP-1/PRP-2/PIF-s and Db-s, showing the additional phosphate group at Ser-17; (ii) the mono-phosphorylated forms at either Ser-22 or Ser-8 of PRP-1/PRP-2/PIF-s, PRP-3/PRP-4/PIF-f, Db-s and Db-f; (iii) a nonphosphorylated form of PRP-3/PRP-4/PIF-f; (iv) the triphosphorylated and diphosphorylated forms of Pa 2-mer. Moreover, minor quantities of PRP-3/PRP-4/PIF-f lacking the C-terminal Arg (11,006 amu), and of Pa 2-mer lacking the C-terminal Gln (30,793 amu) were found. By this approach the different phenotypes of PRH1 locus in 59 different subjects were characterized.     

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive     

A field guide to the proteomics of post-translational modifications in DNA repair

All cells incur DNA damage from exogenous and endogenous sources and possess pathways to detect and repair DNA damage. Post-translational modifications (PTMs), in the past 20 years, have risen to ineluctable importance in the study of the regulation of DNA repair mechanisms. For example, DNA damage response kinases are critical in both the initial sensing of DNA damage as well as in orchestrating downstream activities of DNA repair factors. Mass spectrometry-based proteomics revolutionized the study of the role of PTMs in the DNA damage response and has canonized PTMs as central modulators of nearly all aspects of DNA damage signaling and repair. This review provides a biologist-friendly guide for the mass spectrometry analysis of PTMs in the context of DNA repair and DNA damage responses. We reflect on the current state of proteomics for exploring new mechanisms of PTM-based regulation and outline a roadmap for designing PTM mapping experiments that focus on the DNA repair and DNA damage responses.     

Metal binding characteristics of human salivary and porcine pancreatic amylase

With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.     

质谱图聚类网络法在鉴定多肽翻译后修饰中的应用及研究进展

蛋白质组学多肽鉴定方法一直以基于质谱分析和数据库搜索的方法为主,随着质谱仪技术的发展,海量的质谱数据被获取,这为大规模蛋白质的鉴定提供了一个强大的数据仓库,使得以质谱数据为基础的蛋白质组学研究成为主流。传统的串联质谱图搜库方法鉴定多肽翻译后修饰时具有诸多局限,质谱网络方法可以在一定程度上弥补局限。文中系统综述了基于质谱聚类的质谱网络和质谱图库搜索方法的发展历程、理论研究和应用研究,讨论了质谱网络库方法在鉴定多肽翻译后修饰的优势,并进行了分析和展望。     

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.     

非限制翻译后修饰鉴定方法的研究进展

蛋白质翻译后修饰在真核生物细胞内广泛存在,对蛋白质的结构和功能有着十分重要的影响.串联质谱技术的快速发展为翻译后修饰鉴定提供了高通量、高灵敏度和高分辨率的分析平台,但传统搜索引擎鉴定修饰的方法无法满足数据分析的需求,非限制翻译后修饰鉴定已成为目前蛋白质组修饰分析的重要手段之一.非限制翻译后修饰鉴定不需要在分析前指定修饰类型,可以直接从样品中找出大量已知或未知的修饰,对提高质谱图谱解析率以及揭示蛋白质的生物学功能具有十分重要的意义.本文首先介绍了非限制翻译后修饰鉴定的定义和发展历程,然后从序列匹配和谱图匹配两个方面详细综述了目前非限制翻译后修饰鉴定的主流算法,分析了非限制翻译后修饰鉴定的质量控制问题,最后结合非限制翻译后修饰鉴定的实际应用讨论了修饰鉴定算法的不足和发展方向.     

Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.     

Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase

Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.     

碱性淀粉酶的发酵生产及其应用研究进展

碱性淀粉酶是诸多碱性酶中的一种,指的是其最适稳定及反应pH值在碱性范围内,在碱性环境中可以保持稳定且可高效催化降解淀粉。碱性淀粉酶在纺织、洗涤剂、医药、食品等领域具有广泛应用。利用嗜碱微生物可以生产碱性淀粉酶。以下综述了碱性淀粉酶的发酵生产及其应用的研究进展。     

翻译后修饰可能影响黄瓜花叶病毒2b 蛋白抑制子活性及稳定性

黄瓜花叶病毒 (Cucumber mosaic virus,CMV) 编码的2b蛋白具有RNA沉默抑制子功能,为了研究翻译后修饰对2b功能的影响,利用反向PCR定点突变方法对CMV-Q株系2b蛋白的1个预测的磷酸化位点 (S40) 和2个预测的泛素化/SUMO化位点 (K22,K39) 进行了点突变,同时将点突变体插入植物表达载体。通过农杆菌共注射法对3个2b突变体的抑制子活性进行了分析,结果证明,当S40突变为A (2bS40A) 后,2b抑制局部和系统沉默的活性均大幅降低;当K22突变为R (2bK2     

The logic of protein post-translational modifications (PTMs): Chemistry,mechanisms and evolution of protein regulation through covalent attachments

Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.