蛋白體學研究技術 (Proteomics)

Transcription

1 蛋白體學研究技術 (Proteomics) SCIENTIFIC AMERICAN, INC. April 2002 科學人 2002 六月號 " 蛋白質當家 " 撰文 / 伊澤爾翻譯 潘震澤 - 基因組計畫之後, 輪到蛋白質當家了! 蛋白組學的狂熱人士正爭先恐後地將我們體內蛋白質分門別類, 並想弄清楚它們之間如何建立聯繫 這方面的努力將可能促成更多 更好的藥品 1

2 The Proteome Coined by M.R. Wilkins in 1995, Biotechnol.. Gene Eng. Rev. Vol 13, p Proteome indicates the total proteins expressed by a genome in a cell or tissue. - Proteome, unlike the genome, is not a fixed feature of an organism. - Proteomics is the study of protein expression and function on a genome scale. " Proteomics is one of the most important post-genomic approaches to understanding gene function " Nature, December 1999 " Proteomics will be bigger than Genomics " Red Herring, June 2000 " BIOTECH S NEXT HOLY GRAIL: now, companies are racing to decipher the human protein set " Business Week, Abril

3 The Central Dogma of Life The Lancet (2000) 356:

4 Why Proteomics? - A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival. - It is not necessary a correlation between transcript and protein expression level. - Modifications of proteins can only be determined by proteomic methodologies. - The localization of gene products. - The protein-protein interactions. - Proteins are the most drug targets. ~ Only a protein-based approach will be able to detect these changes and information ~ " Proteins, not genes hold the keys to biology " International Business Communication, February 1999 " Proteins: The business end of biology " BioVenture View, April

5 Main Areas 1. Micro-characterization of protein identities and their post-translational modifications. 2. "Differential display" - proteome comparison. 3. Protein-protein interactions (identification of protein complex). Nature (2000) 405:

6 Major Technologies in Proteomics Two-Dimensional Electrophoresis (2DE) 二維電泳 - IEF strip separation - SDS-PAGE gel separation Mass Spectrometry (MS) 質譜儀 - Protein sequencing Others - Peptide mapping - Automation (ICAT) - 2D Column chromatography Bioinformatics 生物資訊 (MDLC) - Protein database - Protein chips - Database mining - BIACORE (biosensor) - Yeast two hybrid system 6

7 Two-Dimensional Gel Electrophoresis (2DE) protein solubilization 酸性增強 鹼性增強 蛋白質負電荷減弱 蛋白質正電荷減弱 isoelectric focusing Amersham Biosciences 7

8 2DE - Sample Preparation SDS-PAGE by molecular weight 8

9 2DE Detection by Staining Reagent Silver Coomassie blue 9

10 2DE Detection by Staining Reagent Silver Coomassie Sypro Ruby Cy3 Cy3 + Cy5 Cy5 10

11 2DE - Differential Display 11

12 2DE - Image Processing 50% decrease 50% increase 12

13 2D Databases on Internet 13

14 Drawbacks of 2DE 1. Solubility-dependent method - large and membrane proteins are poorly represented because of solubility problems. 2. Limited mass and ph ranges - not comprehensive. 3. Capacity of total proteins - low abundant proteins are poorly detected. 4. Sensitivity of current detection methods. 5. Reproducibility. 6. Lack of automation. Still Two-dimensional gel electrophoresis provides a relatively easy and efficient method to separate thousands of proteins simultaneously. In addition, no other separation methods offer visualization of posttranslationally modified proteins. 14

15 Mass Spectrometers Q-Tof 2 (Micromass( UK) M@LDI (Micromass( UK) ESI-MS/MS MALDI-TOF 15

16 Principle of Mass Spectrometry 質譜儀 (Mass Spectrometers) - 樣品進量 - 樣品離子化 - 質量偵測 - 離子數目偵測 質譜 (Mass Spectrum) - molecular weight( 分子量 ) - fragmentation information( 結構 ) 16

17 Tandem Mass Spectrometry 胺基酸鍵斷裂 Collision-induced dissociation (CID) 胺基酸定序 Sequencing 17

18 Tandem MS-based Protein Sequencing Concentration-dependent process Favor multiple charged species 18

19 Protein Identification - Sequencing 19

20 Sequencing Search Result 20

21 MALDI-TOF Mass Spectrometry Matrix-Assisted Laser Desorption/Ionization Time-of of-flight 21

22 Protein Identification - Peptide Mapping Peptide mass fingerprinting (PMF) Trypsin * * * * * * * 22

23 Protein Identification - Peptide Mapping 23

24 Peptide Mapping Search Result 24

25 Protein Chip technology Protein chips a variety of bait proteins as ELISA Depend on specific and well-characterized antibodies and a number of technical problems. Nature (2000) 405:

26 ICAT technology Quantitation by mass spectrometry - but using limited or no protein separation Quantitative analysis of complex protein mixture using isotope-coded affinity tags (ICAT) Nature biotechnology (1999) 17:

27 Multi-Dimensional technology IEF-NP RP HPLC - Rotofor (BioRad) - 1g of total protein 27

28 Multi-Dimensional Protein Identification technology (MudPIT) MudPIT is another approach which may help alleviate many of the disadvantages associated with two-dimensional gel electrophoresis. MudPIT uses two chromatography steps interfaced back to back in a fused silica capillary. 28

29 Protein-Protein Interactions Nature (2002) 415: baits, 1440 proteins, 232 complexes Nature (2002) 415: baits, 3617 interactions, 1578 proteins Nature (2002) 415:

30 BIACORE Surface Plasmon Resonance Biosensor Incident Light θ Detector E vanescent Field -Real time biomolecular interaction analysis -Non-invasive optical measuring technique -No labeling technique 30

31 Yeast Two-Hybrid System Molecular & Cellular Proteome (2002) 1: BioEssays (1998) 20:1-6. WebGenNet (genome.c.kanazawau.ac.jp/webgen/webgen.html) 31

32 Major Technologies in Proteomics Two-Dimensional Electrophoresis (2DE) 二維電泳 - IEF strip separation - SDS-PAGE gel separation Mass Spectrometry (MS) 質譜儀 - Protein sequencing Others - Peptide mapping - Automation (ICAT) - 2D Column chromatography Bioinformatics 生物資訊 (MDLC) - Protein database - Protein chips - Database mining - BIACORE (biosensor) - Yeast two hybrid system 32

33 Activity-Based Probe (ABP) PNAS (1999) 96:

34 Properties of Proteomics Techniques Current Opinion in Chemical Biology (2002) 6:

35 Properties of Proteomics Techniques Current Opinion in Chemical Biology (2002) 6:

36 Applications of Proteomics Technology Establishing and mining proteome from different species In EMBL-EBI (European Bioinformatics Institutes a part of European Molecular Biology Laboratory), There are currently proteome sets available for 134 proteomes. Proteome analysis is available for 131 of these. /tw.expasy.org/ch2d/2d-index.html 36

37 Applications of Proteomics Technology Drug Target/marker identification This application of proteomics provides a protein profile of a cell or tissue that can be used to compare a healthy with a diseased state for protein differences in the search for drugs or drug targets. This is the most popularized of the applications for proteomics; it has the added unique advantage that bodily fluids can be used for profiling. 1. Body fluids 1.1 Blood cells Erythrocytes Leukocytes Monocytes/macrophages Lymphocytes 2. Solid tissues Platelets 2.1 Heart 1.2 Plasma and serum 2.2 Brain 1.3 Urine 2.3 Thyroid 1.4 Amniotic fluid 2.4 Muscle 1.5 Cerebrospinal fluid 1.6 Synovial fluid 3. Malignant 1.7 Saliva diseases 1.8 Sweat 4. Tissue culture 1.9 Tears 5. Malignant cells 1.10 Semen 6. Bacterial proteins 1.11 Other fluids 37

38 Identification of A Putative Cancer Marker SCC589 Normal Bladder cancer is a multifactorial disease with both an environmental and genetic component. The main contributors to bladder cancer are considered to be tobacco smoking, occupational exposure to hazardous compounds (mainly the carcinogens 4-aminobiphenyl, 2-naphthylamine and benzidine) and the parasite Schistosoma haematobium. - Transitional Cell Carcinoma (TCC) - Squamous Cell Carcinoma (SCC) SCC Electrophoresis (1999) 20: Current status: ELISA kit: 1-5 µg/ml (~ 10-7 M) TCC 38

39 CJD) Creutzfeldt-Jakob disease (CJD) 庫茲德賈克氏病 - 一種會傳染的神經疾病, 具有和瘋牛病同類的病原, 是一種變性的蛋白質, 喜歡侵襲神經系統 A patient with rapidly progressing dementia was found to have a spinal fluid containing two unusual proteins (NO.130, NO.131) which were recently identified as Tau γ chain (14-3-3). Immunoassay against CJD patients: 68 out of 71 was positive. Hsiech et al. N Engl J Med (1996) 335:

40 Red: Animals with clinical dilated cardiomyopathy. Green: Normal animals. Purple: Clinically normal but are offspring of two parents with clinical dilated cardiomyopathy. 40

41 Proteomics & drug mode-of-action studies Fluorouracil (5-FU) is chemotherapy that is given as a treatment for some types of cancer including bowel, breast, stomach, and gullet cancer. Known common side effects: sore mouth and taste change, diarrhea, gritty eyes and blurred vision, skin changes, temporary reduction in the production of blood cells by the bone marrow, tiredness and a general feeling of weakness. OGT719, a novel galactosyl derivatives of 5-FU for treatment of hepatocellular carcinoma and colorectal metastases localized in liver. Among 2291 proteins, 19 proteins which were changed five-fold or more by both drugs, were identified. Strongly suggesting similarities in the mode of action for these two compounds. 41

42 Measurement of Drug Toxicity The measurement of drug toxicity includes studies on mechanistic toxicology for evaluating current drugs and on predictive toxicology for screening new drug candidates. Drug side-effects are common problems but the action mechanisms of the drug toxicity on human organs are largely unknown. In a typical proteomic experiment, protein extracts from a targeted organ after overdose or over-time dosing of a drug are separated by means of 2-DE or protein chips and the differentially expressed proteins are identified and analyzed. Further characterization of these altered proteins helps us understand the mechanism of toxicity for drug re-design and improvement, and the elicited drug-associated proteins can be used as predictive markers of toxicity for classifying compounds and screening large numbers of drug candidates. Overdose of acetaminophen (APAP) causes acute hepatoxicity in rodents and man. Many of the proteins showing changed expression levels are either known targets for covalent modification by N- acetyl-p-benzoquinoneimine (NAPQI) or involved in the regulation of mechanisms that are believed to drive APAP-induced hepatotoxicity. 42

43 Measurement of Drug Screening Studies on drug toxicity may not only elucidate the mechanism of toxic damage but also detect toxin-associated proteins, which can be used as markers of toxicity for drug screening. A full drug-screening program involves the establishment of comprehensive databases integrated with techniques and data from genomics, proteomics, and bioinformatics. 43

44 The Impact on Drug Discovery & Therapy Identification of drug targets Action mechanism of drugs Drug toxicity and screening Lead optimization SCIENTIFIC AMERICAN,, APRIL 2002 p41 44