磷酸化蛋白质组学分析和定量技术的研究进展

蛋白质的磷酸化是一种可逆性的蛋白质翻译后修饰,在生物体内起着极为重要的作用.近年来蛋白质翻译后修饰日益成为蛋白质组研究的热点之一.定量磷酸化蛋白质组学方法和技术的快速发展为研究蛋白质磷酸化时空动态变化,更好地了解生物学功能调节网络奠定了坚实的基础.作为蛋白质组学研究的一个重要组成部分,定量磷酸化蛋白质组学因其磷酸化蛋白质所具有的独特特征,在技术和方法研究方面将面临更为严峻的挑战.综述了磷酸化蛋白质组学定量的一些分析技术和方法的发展现状、优缺点以及未来的发展趋势.     

精子磷酸化蛋白质组学研究应用

蛋白质磷酸化是生物体中广泛存在的翻译后修饰方式,参与多种过程的调节。精子是高度分化的特殊细胞,不具有转录活性,主要依赖于蛋白质的磷酸化完成精子成熟、分化和受精等过程。因此,对于精子磷酸化蛋白质组学的研究有助于进一步了解精子发生、精子获能、超激活以及精卵识别等过程的调控机制。本文简要综述了精子磷酸化蛋白质组学的研究方法及磷酸化蛋白质组学在精子中的应用,为精子磷酸化蛋白质组学在实际科研应用中提供了理论参考。     

磷酸化蛋白质组学的新进展及其在肝脏生理和病理机制中的应用

蛋白质磷酸化是最常见的蛋白质翻译后修饰形式。由于蛋白质的磷酸化形式可以被磷酸酶和磷酸激酶进行可逆的调控,所以在众多的生命活动过程中蛋白质的磷酸化修饰起着重要的调控作用,因此对生物体内蛋白质磷酸化修饰的系统研究对于揭示生命科学的奥秘显得十分重要。近年来,随着质谱技术和生物信息学软件以及磷酸化肽段富集方法的发展,利用质谱对生物体内蛋白质磷酸化修饰研究的技术逐渐成熟。肝脏作为人体最重要的代谢和免疫器官,深入研究肝脏细胞内蛋白质磷酸化修饰形式对于理解其功能具有重要指导意义。目前,迅速发展的磷酸化蛋白质组学技术已经被广泛应用到肝脏功能的生物学研究中。这些研究加深了人们对肝脏的生理及病理状态的分子生物学机制的了解。本文综述了当前磷酸化蛋白质组学的研究进展和磷酸化蛋白质组学在肝脏中的研究。     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

蛋白质修饰组学在肉品质研究中的应用

蛋白质翻译后修饰(Post-translational modifications, PTMs)在生命体中具有十分重要的作用。生命有机体中常见的PTMs有磷酸化、酰化、糖基化、泛素化、乙酰化、氧化和甲基化等。文章主要介绍了蛋白质组学在肉制品科学方面的应用、PTMs的主要内容以及分析蛋白修饰特性常见技术的发展,总结了PTMs对肌肉生理特性的影响和蛋白质组学方法在肉质蛋白质修饰研究中的重要性及前景,讨论了利用蛋白质修饰组学技术研究肌肉熟化过程中品质特性变化的特点。     

蛋白质组学方法在细胞内信号转导研究中的应用

蛋白质组学的新技术为我们研究细胞内的信号转导过程提供了更广泛和崭新的思路,它克服了传统技术的局限性,实现了对蛋白的高通量分析。简要综述了蛋白质组学技术在信号转导过程中信号分子的确定、定量,磷酸化等翻译后修饰的识别,以及蛋白质之间相互作用研究等方面的应用。     

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。     

玉米早期花药蛋白质组和磷酸化蛋白质组分析

蛋白质磷酸化修饰是调控其功能的一种重要方式。植物有性生殖过程在农作物产量形成和物种繁衍过程中起着重要作用。作为植物雄性生殖器官的花药,其正常生长发育对于保证形成功能性配子(花粉)以及完成双受精过程至关重要。本研究以重要农作物玉米(B73)为材料,利用Nano UHPLC-MS/MS质谱技术对玉米早期发育的花药在蛋白质组和磷酸化蛋白质组水平进行全面分析,以探究玉米花药发育过程中的蛋白调控网络和磷酸化修饰调控网络。在蛋白质组学分析中,共鉴定到了3 016个多肽,匹配到1 032个蛋白质上。通过Map Man分析,预测到了一些和花药发育相关的蛋白质,例如受体激酶(GRMZM2G082823_P01、GRMZM5G805485_P01等)。另外,在磷酸化蛋白质组学研究中,通过对Ti O2亲和层析富集到的磷酸化多肽进行质谱分析,检测到了257个磷酸化多肽,匹配到210个蛋白质上。我们的数据揭示了玉米花药发育过程中的223个磷酸化位点。与已发现的玉米中的86个磷酸化蛋白质(植物蛋白磷酸化数据库(P3DB):http://www.p3db.org/organism.php)相比,其中203个磷酸化蛋白和218个磷酸化位点为首次揭示。进一步生物信息学分析表明:磷酸化在14-3-3蛋白质、激酶、磷酸酶、转录因子、细胞周期和染色质结构相关的蛋白质介导的玉米早期花药发育过程中起着重要的调控作用。总之,本研究首次在蛋白质组学和磷酸化蛋白质组学水平研究了玉米早期花药发育的蛋白质调控网络,不仅丰富了玉米蛋白质和磷酸化修饰蛋白质数据库,并为利用遗传学和生物化学手段深入研究玉米花药发育的分子调控机理提供了基础。     

哺乳动物精子磷酸化蛋白质组学研究进展

蛋白质的表达、修饰及相互作用的研究已成为后基因组学时代蛋白质组学中的重要内容。蛋白质磷酸化和去磷酸化作为最普遍的翻译后修饰之一,是精子细胞信号转导和酶调控、表达的主要分子机制,亦是精子、卵细胞信号识别及完成受精作用的关键环节。对精子磷酸化蛋白功能的研究有助于深入理解精子的获能、超激活运动的维持、发生顶体反应及精卵结合等受精过程的分子调控机理。对哺乳动物精子磷酸化蛋白质组学的研究进展,包括动物精子磷酸化蛋白质组学研究的技术方法、磷酸化蛋白质种类的鉴定、定量及其功能分析进行了综述,为进一步发掘与受精相关的重要生物标志物,揭示精子发育、繁殖潜能变化及受精分子机理奠定基础。     

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。     

Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin

We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

     

Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents

Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.     

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO₂) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO₂ and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO₂ particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO₂ particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.     

Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh

The RNA-binding protein 8A (RBM8A)–mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect in vitro and in vivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.     

A method for profiling the phosphorylation state of tyrosine protein kinases

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.     

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.     

Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates

The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading dye solution were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated Shc and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique.     

植物中的MAPK及其在信号传导中的作用

促分裂原活化蛋白激酶(MAPKs)是一类存在于真核生物中的丝氨酸/苏氨酸蛋白激酶。同动物和酵母中MAPKs类似,植物中的MAPK级联途径也是由MAPKs、MAPKKs、MAPKKKs三种类型的激酶组成。植物细胞内受体接受外界刺激信号,然后依次磷酸化激活MAPKKKs、MAPKKs和MAPKs,并影响相关基因表达。目前已经从植物中分离到一些MAPKs、MAPKKs和MAPKKKs,它们参与了植物激素、生物胁迫及非生物胁迫等过程的信号传导。介绍了植物响应外界环境胁迫过程中,不同机制和因子对MAPKs级联途径的调控。     

Xanthomonas campestris sensor kinase HpaS co-opts the orphan response regulator VemR to form a branched two-component system that regulates motility

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.