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。     

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．     

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。     

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。     

Phos-tag技术在磷酸化蛋白质组学中的应用

Phos-tag是新研制出的一种对磷酸基团具有特殊亲和力的化合物。由于其对磷酸化蛋白质具有高特异性、高亲和力等特点使其迅速在磷酸化蛋白质的检测、分离和纯化等方面得到广泛的应用。本文综述了Phos-tag的化学性质、原理及其近年来在磷酸化蛋白质组学中的应用,并与传统的磷酸化蛋白质组学研究技术做了比较,对未来磷酸化蛋白质组学的研究技术作了展望。     

定量蛋白质组学研究技术

随着蛋白质组研究的深入发展,人们已不满足对一个混合体系中蛋白质进行定性和简单定量分析,要求更加准确的定量分析。为此,有人提出了“定量蛋白质组学”概念。目前,应用于定量蛋白质学的研究技术主要有：蛋白质荧光染色技术,同位素标记技术,同位素亲和标签技术,蛋白质芯片技术。     

定量蛋白质组学分析PknG在分枝杆菌中的功能

丝氨酸苏氨酸蛋白激酶G(PknG)是分枝杆菌中一个类似于真核生物蛋白激酶C的蛋白质,对结核分枝杆菌的生长和新陈代谢等生理过程,以及结核分枝杆菌的耐药和在宿主细胞中的存活都起着重要的调节作用.本文在耻垢分枝杆菌(Mycobacterium smegmatis)mc2155中构建了过表达结核分枝杆菌PknG的重组菌株PknG-mc2155,并发现PknG-mc2155的生长速度慢于mc2155.应用化学修饰结合LC-LC-MS/MS的定量蛋白质组学方法,在mc2155和PknG-mc2155中鉴定到了176种有差异表达的蛋白,其中152种蛋白在PknG-mc2155中表达下调,24种蛋白表达上调.这些差异表达的蛋白参与了多个细胞过程,包括代谢、蛋白翻译等.基于这些结果,我们推测PknG-mc2155生长速度慢的原因是因为代谢相关酶如GlpK,ALD和DesA1等蛋白表达的下调;而Ag85A,Ag85C,SecA2等蛋白的上调则增强细菌的感染性;另外KatG蛋白的下调提示PknG的过表达增强了菌株的抗药性.代谢组学分析发现谷氨酸和谷氨酰胺在PknG-mc2155中的水平低于在mc2155中水平,证实了PknG影响谷氨酰胺的稳态平衡.利用蛋白质磷酸化分析,我们发现PknG的苏氨酸残基T-320上有一个自磷酸化修饰,而且在PknG-mc2155菌株中,也鉴定到gltA和glmM上的磷酸化修饰,显示gltA和glmM是PknG的底物.本研究为理解PknG的功能和作用机制提供了新的依据和解释,为深入研究PknG在结核分枝杆菌中的功能奠定了基础,我们的结果也表明蛋白质组学技术是系统研究细菌蛋白质功能的重要工具.     

基于质谱的定量蛋白质组学策略和方法研究进展

定量蛋白质组学已经成为组学领域研究的热点之一.相关实验技术和计算方法的不断创新极大地促进了定量蛋白质组学的飞速发展.常用的定量蛋白质组学策略按照是否需要稳定同位素标记可以分为无标定量和有标定量两大类.每类策略又产生了众多定量方法和工具,它们一方面推动了定量蛋白质组学的深入发展;另一方面,也在实验策略与技术的发展过程中不断更新.因此对这些定量实验策略和方法进行系统总结和归纳将有助于定量蛋白质组学的研究.本文主要从方法学角度全面归纳了目前定量蛋白质组学研究的相关策略和算法,详述了无标定量和有标定量的具体算法流程并比较了各自特点,还对以研究蛋白质绝对丰度为目标的绝对定量算法进行了总结,列举了常用的定量软件和工具,最后概述了定量结果的质量控制方法,对定量蛋白质组学方法发展的前景进行了展望.     

磷酸化蛋白质组学在三阴性乳腺癌诊治研究中的进展

三阴性乳腺癌是乳腺癌中恶性程度最高的亚型,其治疗仍以化疗为主,但容易出现耐药,且患者预后较差。随着蛋白质组学技术的发展,磷酸化蛋白质组学研究取得了长足的进步,并在肿瘤发生发展机制和诊治研究中得到了广泛的应用。同样,磷酸化蛋白质组学在三阴性乳腺癌的发生发展、靶向治疗和耐药机制研究等方面也发挥着重要作用。本文主要对目前磷酸化蛋白质组学在三阴性乳腺癌中的研究进展进行综述,旨在为基于磷酸化蛋白质组学的三阴性乳腺癌发生发展机制和诊治研究提供指导和帮助。     

同位素标记相对和绝对定量技术研究进展

定量蛋白质组学是蛋白质研究的前沿学科。目前常用的定量蛋白质组学研究技术有荧光差异凝胶电泳（DIGE）、同位素亲和标记（ICAT）等。同位素标记相对和绝对定量（iTRAQ）技术是近年来最新开发的一种新的蛋白质组学定量研究技术。结合非凝胶串联质谱技术,该技术可对复杂样本、细胞器、细胞裂解液等样本进行相对和绝对定量研究,具有较好的定量效果、较高的重复性,并可对多达四种不同样本同时进行定量分析。本文对 iTRAQ 技术的原理、实验方法及应用进展进行了综述。     

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。     

Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling

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Phosphoproteomics in analyzing signaling pathways

A surprisingly fewer than expected number of genes in the human genome suggests that sophistication of its biologic system is, in part, due to complex regulation of protein activities. The activities of most cellular proteins are regulated by post-translational modifications. One of the most important post-translational modifications is reversible protein phosphorylation, which decorates more than 30% of the proteome and regulates signal transduction pathways under normal conditions as well as in disorders such as diabetes, neurodegenerative diseases, autoimmune diseases and several forms of cancers. This review examines the recent developments in mass spectrometry-based methods for phosphoproteome analysis and its applications for the study of signal transduction pathways. The basic principles of non-mass spectrometry-based methods, such as chemical genetics and flow cytometry-based approaches, are also discussed as well as their specific advantages to signaling studies. Finally, signaling pathways are discussed in the light of large-scale protein interaction studies. The proteomic methods addressed in this review are emerging as some of the essential components in systems biology, which seeks to describe signaling networks through integration of diverse types of data and, in the future, to allow computational simulations of complex biologic pathways in health and disease.     

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.     

Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator

Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems‐level perturbations mediated by PP2A activation, we carried out mass spectrometry‐based phosphoproteomic analysis of two KRAS mutated non‐small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS‐related signaling are attenuated for A549 compared to H358, while the effects on cell cycle‐related kinases are noticeably more prominent in A549. Network‐based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics reveals novel modes of function and inter‐relationships among PIKKs in response to genotoxic stress

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3‐kinase‐related protein kinases (PIKKs): ATM, ATR, and DNA‐PK. ATM is missing or inactivated in the genome instability syndrome, ataxia‐telangiectasia (A‐T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild‐type and A‐T cells treated with the double‐strand break‐inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine‐tuned dynamics between the PIKKs following genotoxic stress, such as DNA‐PK‐dependent attenuation of ATM. In A‐T cells, partial compensation for ATM absence was provided by ATR and DNA‐PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.