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1
Introduction to Proteome and the Proteomic
蛋白質體及蛋白質體學介紹
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Desucing the proteome from the genome
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600種甚至上千種的新陳代謝反應,約4萬個基因,10 ~ 15萬個拼接變異種 (splice variants) mRNA,50 ~ 200萬種不同的蛋白質型態 (包括轉譯後的各種修飾),各種分子之間的交互作用所架構起來的細胞內外線路更是不計其數
「體學」的概念(或稱「組學」Omics)
研究細胞、組織或是整個生物體內某種分子 (DNA、RNA、蛋白質、代謝物或其它分子) 的所有組成內容,就稱為「體學」
魏斯坦醫師: 美國國家癌症研究院 (NCI) 分子藥理學實驗室主持人
Genomics (基因體學)Proteomics (蛋白質體學)Transcriptomics (轉錄體學)Kinomics (動力體學)CHOmics (醣類體學)Immunomics (免疫體學)Toxicomics (毒理體學)Metabonomics/Metabolomics (代謝體學)Clinomics (臨床體學) Functional Genomics (功能基因體學)Structural Genomics (結構基因體學)Pharmacogenomics (藥理基因體學)Pharmacoproteomics (藥理蛋白質體學)Ecomics (生態體學=Ecogenomics+Ecoproteomics)Pharmacomethylomics (藥理甲基化體學)
「體學」的概念(或稱「組學」Omics)
Genome (基因體): entire sets of materials in the chromosomes (DNA).
DNA sequencing
http://www.ncbi.nlm.nih.gov/Human Genome Project
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Proteome: a new word, a new field of biology
PROTEins expressed by a genOME
2-D electrophoresis
Post Separation
analysis
Post TranslationalModification
Bioinformatics
Discovery
基因定序基因晶片
二維電泳
蛋白質晶片
(1) Proteome:研究蛋白質之表現 (二維電泳、MALDI-TOF MS)
(2) Interactome:研究蛋白質的交互作用(二維電泳、MALDI-TOF MS)
(3) Phosphoproteome:研究蛋白質的 1D 結構(序列) (MALDI MS/MS)
(4) Glycoproteome:研究醣蛋白質之 2D-3D 結構 (Ion-trap MS)
(5) Structural proteome:研究蛋白質的 3D 結構 (x-ray 繞射)
Genome (DNA) Transcriptome (RNA) Proteome (Protein)
2D electrophoresis
第一維:Isoelectric focussing electrophoresis
2D electrophoresis (二維電泳)
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Carrier Ampholytes
Carrier Ampholytes
Carrier Ampholytes Batch-to-batch variation Immobiline (immobilized pH gradients)
Immobiline (immobilized pH gradients) Immobiline
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Immobiline 1-D SDS PAGE 2-D SDS PAGE
Image Scanning
Visible light: silver staining
Fluorescence: Sypro Ruby Image Analysis Software: PDQuest, Image Master, Melanie
Hepatoma (GP7TB) Normal Hepatocyte (WB)
up-expressiondown-expression
2D electrophoresos, in-gel digestion and MALDI-TOF
trypsin
Trypsin
Robotic System for Automatic Proteomics
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MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight)
E=Ezes = (1/2) mv2
MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight)
Ez = (1/2) mv2
MALDI-TOF with Delayed Ion Extraction
MALDI-TOF with Delayed Ion Extraction MALDI-TOF with Reflectron
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MALDI-TOF with Reflectron
Trypsin digestion
protein database
Peptide fingerprint
Peptide Mass Fingerprint (PMF)
Rat Tubulin Beta Chain (matched peptides: 8)Mw: 49.9 pI: 4.8
Advantages of MALDI-TOF and peptide fingerprint1. Bioinformatics (MS-fit, MS-tag):
rapid identification of protein by searching protein and translated cDNA database.
2. Identification of proteins in large scale without using any antibodies !!!!!
1D electrophoresisWestern blotting
220
97.4
66
46
30
2D electrophoresisMALDI-TOF
Disadvantages of MALDI-TOF and peptide fingerprint
1. Peptide fingerprinting does not provide sequence information of peptides.2. Bioinformatics
Without complete sequence information, we cannot find novel genes fromgenome database by BLAST.
De novo sequencing by tandem mass spectrometry (MS-MS)
1. Various types of tandem MSTriple quadrupole, Q-TOF, TOF-TOF, Ion-trap
2. Collision-induced dissociation(CID)
Ar
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Peptide Fragmentation and Ionization
Ar (CID)
Tandem Mass Spectrum
Search protein database2D gel In-gel digestion and MALDI-TOF
Search DNA databaseMS-MS
Sequence 1 Sequence 2…...
Search EST or genome database for obtaining novel genes
Design oligonucleotide probefor gene cloning from cDNAlibrary
Analytical strategy
peptidefingerprint
BLASTDegenerateDNA sequence
Phosphoproteomics(Identification of all phosphorylatedproteins)
Identification of Phosphorylated Proteins
Western blotting
(Anti-phosphoamino acid Ab)
Exp. 2
Exp. 1
TSTEPQBQPGENLB=phosphotyrosine b7-b6 = 243
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0
100
%
MSMS OF 1543 DHB 26 (0.504) AM (Cen,2, 50.00, Ar,5000.0,0.00,0.30); Sm (Mn, 1x2.00); Cm (2:27) TOF MSMS 1543.00ES+ 4.01e3b8
1015.3774
y''5529.2648
PQBBQP
469.1490
QPPQ
EN~226.1226
187.0850155.1096284.1253 398.1670
PQBQQBQP
597.2139
997.3692
y''6657.3303
979.3594b7887.3309
658.3300
QBQPGEN^869.3159
809.3040659.3278
904.3628
y''91125.4500
1016.3760
1017.3768
1018.3921
1126.4546
1544.5708
1543.5778c9
1127.45581543.5295
1526.5491b111298.4583
1128.4614 y''101254.4733
1170.4174
1299.4668 b121412.4886
1300.4686 1508.54691507.5514
1545.5356
1546.5725
1547.5634
1548.6073
b6 644.29
Phosphorylation sites mapping
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Interactomics(Identification of all protein-proteininteractions)
Peptide tag
pEBG (GST)pEBH (polyhistidine)pMEF (FLAG)
Transfection into 293T cells
Over-expression of tagged proteinCell lysis
Coimmunoprecipitation
Protein-protein interaction (coimmunoprecipitation, GST pull-down)
Bait protein