题目:蛋白质磷酸化研究
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题目:蛋白质磷酸化研究. 汇报人:王顺 . 2013.05.09. 1. 汇报提纲. 一、蛋白质磷酸化 二、磷酸化蛋白质分析样品的富集及制备 三、蛋白分离后的检测、鉴定及其磷酸化位 点的预测 四、文献. 2. 一、蛋白质磷酸化. 蛋白质磷酸化:指由蛋白质激酶催化的把ATP或GTP γ位的磷酸基转移到底物蛋白质氨基酸残基上的过程。. 3. 蛋白质磷酸发生位点. 真核生物,丝氮酸、苏氨酸、酪氨酸等 原核生物,天冬氨酸、谷氨酸、组氨酸等. 有些蛋白质在二者中均可被磷酸化,它们的磷酸化位点通常是精氨酸、赖氨酸和半胱氨酸残基。. 4. 磷酸化蛋白质分类.

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题目:蛋白质磷酸化研究

汇报人:王顺 

2013.05.09

1


汇报提纲

一、蛋白质磷酸化

二、磷酸化蛋白质分析样品的富集及制备

三、蛋白分离后的检测、鉴定及其磷酸化位 点的预测

四、文献

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一、蛋白质磷酸化

蛋白质磷酸化:指由蛋白质激酶催化的把ATP或GTPγ位的磷酸基转移到底物蛋白质氨基酸残基上的过程。

3


蛋白质磷酸发生位点

真核生物,丝氮酸、苏氨酸、酪氨酸等

原核生物,天冬氨酸、谷氨酸、组氨酸等

有些蛋白质在二者中均可被磷酸化,它们的磷酸化位点通常是精氨酸、赖氨酸和半胱氨酸残基。

4


磷酸化蛋白质分类

O-磷酸盐蛋白质

N-磷酸盐蛋白质

酰基磷酸盐蛋白质

S-磷酸盐蛋白质

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蛋白质磷酸化的功能

①磷酸化参与酶作用机制,此过程磷酸化为反应中间产物(多为S-或N-磷酸盐)。

②磷酸化介导蛋白活性,蛋白分子通过蛋白激酶发生磷酸化而改变性能。

③磷酸化发挥各种独特的生理效应,如天冬氮酸、谷氨酸和组氨酸的磷酸化在细菌趋化反应的感觉性传导中发生解离。

④在信号转导中两个主要方面的作用:(1)通过磷酸化调节蛋白活性;(2)通过蛋白逐级磷酸化使传导信号逐级放大,进而引起细胞反应(生理生化活动)。

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二、磷酸化蛋白质分析样品的富集及制备

富集磷酸化蛋白质的方法:

生物素亲和素富集;

金属离子亲和层析(IMAC);

免疫沉淀;

磷酸化抗体亲和层析等。

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生物素亲和素富集:

第一种方法是在强碱性环境中加入硫基乙醇,替换丝氨酸和苏氨酸残基上的磷酸基团,使巯基暴露并与生物素亲和标签相结合,然后通过链霉素包被的磁珠将磷酸化蛋白或磷酸肽从复合体中分离出来。胱氨酸和甲硫氮酸也可以这种方法进行衍生。

第二种方法是通过碳二酰乙胺浓缩反应将胱胺加到磷酸基团上,再通过碘乙酰磁珠对磷酸化蛋白或磷酸肽进行亲和分离。

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金属离子亲和层析法:磷酸基团与固相化的金属离子有高亲和力,可被选择性地吸附在上面,富集微量磷酸肽样品,可以进行金属离子亲和层析法:磷酸基团与固相化的金属离子有高亲和力,可被选择性地吸附在上面,富集微量磷酸肽样品,可以进行IMAC纯化的磷酸肽的离线分析与在线的电喷雾质谱分析。由于IMAC柱会与其他带负电的氨基酸如天冬氨酸和谷氨酸等相结合,层析之前应对蛋白样品中所有的羧酸基团进行甲酯化修饰。

利用抗体与磷酸化蛋白特异结合是最简单的富集方法,高亲和性抗体可以从复杂混合物中免疫沉淀特定的蛋白。

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三、蛋白分离后的检测、鉴定及其磷酸化位点的预测

用32P选择性标记磷酸化蛋白

1、通过纯化激酶及[γ-32P]进行体外标记

2、利用[γ-32P]-ATP或32PO43- (正磷酸盐)平衡胞内的ATP水平进行体内标记

然后经分离得到。

埃德曼降解测序

磷酸化位点分析的标准途径是用印记标记纯化的磷酸化蛋白,通过一向的薄层电泳和二维的薄层层析(被称为二维肽谱)对所得到的肽段进行分离,放射自显影检测磷酸化肽段,进行埃德曼降解测序;或使用荧光标记磷酸肽。然后通过磷酸化残基特定的滞留时间、荧光标记或放射性标记的氨基酸的释放最终定位磷酸化位点释放的氨基酸进行鉴定。

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质谱技术

定义:质谱法就是通过电场和磁场将带电荷运动粒子(包括原子、分子,甚至分子结构碎片等)按其质荷比分离后进行物化性质检测分析的方法。

原理:一是与非磷酸基团修饰的肽段相比,单磷酸基团修饰的肽段在相对分子质量上增加了79.983;二是磷酸肽还将产生一个可用于诊断的片段离子,而非磷酸化修饰的肽则没有这种现象。

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片段离子分析

多肽骨架片段化技术,能使肽链序列重构,如果序列中包含磷酸化残基,则可以对其进行明确定位。

电喷雾离子源在负电模式下激发带有负电的磷酸基团,进行串联质谱分析可以得到特异的磷酸化片段离子。碰撞诱导解离(CID)用于产生片段离子,所得到的片段离子再通过各种质谱进行分析,从而鉴定磷酸肽。

CID谱的解释是一个复杂的过程,通过对应于一套相互重叠的肽的片段离子的鉴定从而构建出其完整序列,计算组成性离子之间的相对分子质量差别,并将该差别与标准的氨基酸相对分子质量进行比较。

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质谱技术举例

ESI-MS质谱:通过测量样品组份的质荷比(M/Z), 检测样品组份的分子量,对多肽、蛋白质和寡核苷酸等物质定性、定量并能与高效液相色谱仪联用对混合物进行分析。

特点:

对于高分子化合物的测定由于可以产生多电荷峰,与传统的质谱相比扩大了检测的分子质量范围,同时提高了仪器的灵敏度。

ESI-MS是一种软的电离方式,在一定的电压下它不会使样品分子产生碎片,因此对于小分子的样品ESI谱图可确定样品的组成成份有几种。但对于大分子的蛋白质来说由于要形成非常复杂的多电荷峰,因此分析大分子混合物较为困难,一般只用于分析较纯的大分子化合物。

13


质谱技术举例

基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱:MALDI原理是用激光照射样品与基质形成的共结晶薄膜,基质从激光中吸收能量传递给生物分子,而电离过程中将质子转移到生物分子或从生物分子得到质子,而使生物分子电离的过程。TOF的原理是离子在电场作用下加速飞过飞行管道,根据到达检测器的飞行时间不同而被检测即测定离子的质荷比(m/z)与离子的飞行时间成正比,检测离子。具有操作简单、快速、谱图直观、能耐受一定浓度的盐和去垢剂等特点,特别适合于混合多肽、蛋白、寡核苷酸的精确质量数测定。

14


质谱技术举例

傅里叶变换回旋共振质谱(FI-ICR MS):通过用亚热电子轰击离子流来获取含磷酸基的离子和磷酸肽片段。

利用电子捕获解离(ECD)技术,可以轻松打开二硫键,而易断的翻译后修饰的共价键却可以在ECD破碎肽链时保存下来,因而适于翻译后修饰的研究,所得到的离子谱系比CID的更容易解释。

15


质谱技术举例

气质联用(GC-MS):它以气相色谱作为分离系统,质谱为检测系统。样品在质谱部分和流动相分离,被离子化后,经质谱的质量分析器将离子碎片按质量数分开,经检测器得到质谱图。适合于低分子化合物(分子量<1000)分析,尤其适合于挥发性成分的分析。

液质联用(LC-MS):它可以分析气相色谱-质谱(GC-MS)所不能分析的强极性、难挥发、热不稳定性的化合物之外

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Analysis of the yeast phosphoproteome

endo-Lys C as the proteolytic enzyme;

immobilized metal affinity chromatography for phosphopeptide enrichment;

nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection;

gas phase ion/ion chemistry,electron transfer dissociation for peptide fragmentation;

Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites.

18



With this approach, we identify 1,252 phosphorylation sites on 629 (10% of the proteome)proteins in a single experiment with 30 g (600 pmols) of protein from a yeast whole cell lysate. We find that the identified phosphoproteins are encoded by a sample of genes that is representative of a wide variety of cellular processes. Expression levels for the identified phosphoproteins range from 50 to 1,200,000 copies per cell.

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We analyze the identified phosphoproteins in the context of interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.

21


22 interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.


23 interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.


24 interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.


( interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.B) A comparison of phosphoprotein interactions to those of random genomic samples. Clique interactions represent genetic or physical interactions between phosphoproteins (or within random subsamples), and total interactions contain all known genetic or physical interactions between phosphoproteins/sampled proteins and the yeast genome.

(C)Arepresentation of the number of model organisms (A. gossypi, C. elegans, D. melanogaster, H. sapiens, and A. thaliana) across which yeast proteins are conserved with significant BLASTP hits. Phosphoproteins are much more likely

than a random yeast protein to be conserved (leftmost bars), and conserved phosphoproteins are much more likely to be conserved in all five genomes examined (rightmost bars). Conservation in just one genome is largely explained by the data from the closest organism to S. cerevisiae, A. Gossypi (overlay in darker colors). Error bars represent 1 standard deviation.

25


( interaction networks and find that they have a significantly higher number of interactions than expected and interact with other phosphoproteins more than expected. We note that the observed phosphoproteins, but not individual phosphosites, are likely to be conserved across very large evolutionary distances.A) A subset of the KEGG sce04110 cell cycle pathway. Proteins hosphorylated in this study appear as bold nodes. Known physical interactions are represented by blue edges, and known genetic interactions are shown as red edges.

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with the use of proteome chip technology, they identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles.

Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast.

27


To develop a kinase-substrate map for eukaryotes, we determined the substrates recognized by 87 different yeast protein kinases, by using a yeast proteome array and the scheme depicted in Fig. a.

a, Overall scheme to identify kinase substrates. Each kinase was over expressed, purified and assayed on protein chips containing about 4,400 proteins spotted in duplicate.

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The 4,200 different protein-kinase–substrate phosphorylations have been assembled into an in vitro phosphorylation network.

a, A map showing the

connections between kinases and substrates. In all, 87 different kinases

(red dots) and 1,325 substrates (blue dots) are represented in the map.

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b, Global localization data can be used to identify only those phosphorylation events occurring between proteins of the same cellular compartment.

c, Functional data can be used to identify substrates with similar functions to those of the kinases phosphorylating them.

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