磷酸化组氨酸研究进展

蛋白质磷酸化是生物体内一种广泛存在的蛋白质翻译后修饰形式,这种氨基酸与磷酸基团共价连接的修饰模式对蛋白质结构和功能起到了重要调节作用.目前天然蛋白质中发现的可磷酸化位点主要有9种氨基酸残基,其中包括以磷酰胺连接的磷酸化组氨酸.虽然该磷酸化形式在原核生物与真核生物中都起到了重要的调节作用,但对于其生物学功能的研究长期存在技术困难.由于磷酸化组氨酸本身不同于其他磷酸化氨基酸的化学性质,如存在异构体、化学不稳定等,其在传统的研究方法中容易发生水解去磷酸化.随着现代生物化学与分子生物学技术的不断进步,人们针对含有磷酸化组氨酸的蛋白质构建了新的制备、分离与表征策略,本领域也因此开始迅速发展.本文从磷酸化组氨酸的化学结构入手,分析其两种异构体的主要理化性质与化学反应特性,并概述了基于此发展的新型化学生物学研究手段以及对于磷酸化组氨酸生物功能的研究进展.     

蛋白质组氨酸磷酸化研究进展

摘要：蛋白质磷酸化是一种可逆的翻译后修饰,这种翻译后修饰可以改变蛋白质的构象,进而使蛋白质活化或者失活。组氨酸磷酸化在细胞信号传导过程中发挥着重要作用,且组氨酸磷酸化与人类某些疾病密切相关,然而,由于组氨酸磷酸化含有P-N键,具备不稳定性,有关于组氨酸磷酸化的报道远远少于其它磷酸化的报道。本综述系统的总结了组氨酸磷酸化在生物学过程中的作用,以及近些年取得的重要研究进展,以期对深入研究组氨酸磷酸化提供理论参考。     

磷酸化蛋白质组学研究中磷酸化肽段富集与分离方法研究进展

对磷酸化蛋白质组（phosphoproteome）进行系统深入的研究依赖于高重复性和特异性的磷酸化肽段富集与分离方法。目前发展了多种不同原理的磷酸化肽段富集方法,它们往往具有不同的选择性和特异性,因此,根据不同的研究目的选择最适合的富集方法显得尤为重要。本文综述了基于亲和色谱法（affinity chromatography）、免疫沉淀法（immunoprecipitation）、化学衍生法（chemical derivatization）、色谱法（chromatography）和其他新发展方法的磷酸化肽段富集方法,详细介绍了各自的优缺点及相关的优化与改进策略。此外,还简单介绍了磷酸化肽段富集与预分方法的不同组合的研究进展。     

蛋白质磷酸化修饰的研究进展

蛋白质磷酸化是最常见、最重要的一种蛋白质翻译后修饰方式,它参与和调控生物体内的许多生命活动。通过蛋白质的磷酸化与去磷酸化,调控信号转导、基因表达、细胞周期等诸多细胞过程。随着蛋白质组学技术的发展和应用,蛋白质磷酸化的研究越来越受到广泛的重视。我们介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白质分析样品的富集及制备、磷酸化蛋白的鉴定及磷酸化位点的预测、蛋白分离后磷酸化蛋白的检测,及蛋白质磷酸化的分子机制,并综述了近年来国内外的主要相关研究进展。     

利用磺基异硫氰酸苯酯化学辅助方法鉴定磷酸化肽修饰位点

利用MALDI-OF质谱结合磺基异硫氰酸苯酯(SPITC)化学辅助的从头测序（de novo sequencing）方法,将用固相金属离子亲和色谱(immobilized metal affinity chromatography,ＩＭＡＣ) 选择性地从混合物中亲和提取的磷酸肽进行磷酸化位点测定,该方法只有肽键断裂产生的带C端的碎片离子系列（y^＋ 离子系列）出现在质谱图中,图谱背景清晰,信噪比高,单纯的y^＋ 片段离子系列使得谱图解析变得非常容易,对于多磷酸化肽的磷酸化位点,不需借助于任何软件,只需简单地计算两峰之间的分子量之差即可确定.     

金属氧化物在磷酸化蛋白质组学研究中的应用

磷酸化肽段的高效富集是磷酸化蛋白质组学中的重要任务,通过综述纳米金属氧化物在磷酸化蛋白组学中的应用,了解磷酸化蛋白组学研究中常用的磷酸化肽段富集材料和方法.该文介绍纳米金属氧化物、核壳结构纳米磁性材料以及金属离子在磷酸化蛋白组学中的富集效果.纳米金属氧化物材料由于其特殊理化性质在富集磷酸化肽段研究中具有特异性强、选择性好特点,在磷酸化蛋白组学研究中得到了广泛应用.虽然纳米金属氧化物在磷酸化蛋白组学的研究中仍然处于起步阶段,可重复性和全面富集仍面临挑战,但是在未来的亚细胞蛋白组学和功能蛋白组学研究中,纳米金属氧化物及其衍生物仍然会起着重要作用.     

用蛋白质组学方法研究蛋白质酪氨酸磷酸化

蛋白质的磷酸化与去磷酸化过程是生物体内普遍存在的信息传导调节方式,几乎涉及所有的生理及病理过程,其中酪氨酸残基的磷酸化作为较高级的进化形式和复杂的多细胞生命的特征表现得尤为突出和重要。但目前对酪氨酸磷酸化缺乏大规模和系统性的研究,近年发展起来的蛋白质组学为细胞和组织中的酪氨酸磷酸化蛋白质的系统研究提供了必要的技术。     

基于质谱的磷酸化蛋白质组学: 富集、检测、鉴定和定量

磷酸化是一种调控生命活动的重要翻译后修饰,调控生物的生长发育、信号转导、以及疾病的发生发展．从上世纪80年代开始,质谱应用于蛋白质磷酸化的检测中,极大地推动了磷酸化蛋白质组学的发展．质谱检测拥有高灵敏度、高通量的特点,更重要的是具有位点分辨率,因此基于质谱的磷酸化蛋白质组检测方法得到不断的发展和推广．常见的磷酸化蛋白质组研究,首先对磷酸化肽段进行富集,然后进行串联质谱分析,最后通过搜索引擎对修饰位点进行鉴定和定量．本文从这个三个基本方面,对磷酸化蛋白质组研究进行综述,并对未来研究发展方向进行讨论．     

用蛋白质组学方法解析磷酸化蛋白质

蛋白质磷酸化和去磷酸化这一可逆过程参与了高等真核生物细胞信号转导、细胞分化和细胞生长等重要过程,并与许多疾病、肿瘤的发生密切相关。蛋白质组学技术的不断发展和完善,可以更好、更多地识别和鉴定磷酸化蛋白质,为解析磷酸化蛋白质提供了可能。章综述了用于分离和鉴定磷酸化蛋白质的蛋白质组学方法。     

植物蛋白质磷酸化的研究技术

本文介绍植物蛋白质磷酸化研究的技术及其应用情况,并对这些技术的应用前景作了展望。     

Characterization of early autophagy signaling by quantitative phosphoproteomics

Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.     

Recent findings and technological advances in phosphoproteomics for cells and tissues

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.     

Sperm phosphoproteomics: historical perspectives and current methodologies

Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm.     

Challenges in plasma membrane phosphoproteomics

The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.     

磷酸化蛋白质组研究中的富集策略

蛋白质的磷酸化与去磷酸化过程,调控着包括信号转换、基因表达、细胞周期等诸多细胞过程。因此,对蛋白质磷酸化修饰的分析是蛋白质组研究中的重要内容。但由于磷酸化蛋白的丰度较低,难以用质谱直接检测。为了解决这个问题,改善质谱对磷酸肽的信号响应,需要对磷酸化蛋白质或磷酸肽进行富集。目前主要的富集方法包括免疫沉淀、固相金属离子亲和色谱、金属氧化物/氢氧化物亲和色谱等。     

The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.     

Synthesis and characterization of histidine-phosphorylated peptides.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.     

Phosphoproteomics by mass spectrometry: insights,implications, applications and limitations

Phosphorylation of proteins is a predominant, reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective, we discuss four aspects of phosphoproteomics: the insights and implications from recently published phosphoproteomic studies and the applications and limitations of current phosphoproteomic strategies. Since approximately 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis-based investigations. Finally, we discuss strategies to measure the stoichiometry of phosphorylation in a proteome-wide manner that is not provided by current phosphoproteomic approaches.     

酪氨酸磷酸化蛋白质组学技术研究进展及其在生物医学研究中的应用

在酪氨酸磷酸化蛋白质组学的研究过程中,酪氨酸磷酸化位点的富集是最重要的一步。目前常用的富集方法是抗体亲和富集或SH2 superbinder富集。此外,通过质谱与生物信息学等技术,可实现大规模酪氨酸磷酸化位点的鉴定。对酪氨酸磷酸化蛋白质组学进行深度覆盖研究,揭示癌症发生发展过程中失调的激酶,将有助于深入理解癌症的发生发展过程;且由于75%的致癌基因是酪氨酸激酶基因,酪氨酸激酶抑制剂作为抗癌药物受到了越来越多的关注。应用酪氨酸磷酸化蛋白质组学技术,可以鉴定与癌症等重大疾病相关的酪氨酸激酶,从而帮助找到酪氨酸激酶抑制剂。总之,酪氨酸磷酸化蛋白质组学技术可以在酪氨酸激酶鉴定、酪氨酸激酶抑制剂研究及酪氨酸磷酸化信号通路研究等生物医学领域中得到很好的应用。     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.