1

Introduction to Proteome and the Proteomic

蛋白質體及蛋白質體學介紹

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3

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