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题目:蛋白质磷酸化研究 PowerPoint PPT Presentation


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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

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2013.05.09

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ATPGTP

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O-

N-

S-

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(S-N-)

(1)(2)()

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(IMAC)

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IMACIMAC

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32P

1[-32P]

2[-32P]-ATP32PO43- ()ATP

()

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()

79.983

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(CID)

CID

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ESI-MSM/Z

ESI-MSESI

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MALDI-TOFMALDITOFm/z

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(FI-ICR MS)

(ECDECDCID

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(GC-MS)<1000

(LC-MS)-GC-MS

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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.

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With this approach, we identify 1,252 phosphorylation sites on 629 10% of the proteomeproteins 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.

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(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.

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(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.

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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-kinasesubstrate 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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