蛋白體學研究技術 proteomics) - ym.edu.tw
TRANSCRIPT
蛋白體學研究技術蛋白體學研究技術 ((Proteomics)Proteomics)SCIENTIFIC AMERICAN, INC.SCIENTIFIC AMERICAN, INC.April 2002April 2002
科學人科學人 2002 2002 六月號六月號
""蛋白質當家蛋白質當家""撰文/伊澤爾 翻譯╱潘震澤撰文/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。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。這方面的努力將可能促成更多、更好的藥品。
1
The ProteomeThe Proteome
-- ProteomeProteome indicates the total proteins expressed by a genome in a cell indicates the total proteins expressed by a genome in a cell or tissue.or tissue.
-- Proteome, unlike the genome, is not a fixed feature of an organiProteome, unlike the genome, is not a fixed feature of an organism. sm. -- Proteomics is the study of protein expression and function on a Proteomics is the study of protein expression and function on a
genome scale.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 2000
Coined by M.R. Wilkins in 1995, Coined by M.R. Wilkins in 1995, BiotechnolBiotechnol. Gene Eng. Rev.. Gene Eng. Rev. VolVol 13, 13, p19p19--50.50.
2
The Central Dogma of Life
The Lancet The Lancet (2000) 356:1749 (2000) 356:1749 --1756.1756.
3
Why Proteomics?-- A cell is normally dependent upon multitude of metabolic and A cell is normally dependent upon multitude of metabolic and
regulatory pathways for its survival.regulatory pathways for its survival.-- It is not necessary a correlation between transcript and proteinIt is not necessary a correlation between transcript and protein
expression level. expression level. -- Modifications of proteins can only be determined by proteomic Modifications of proteins can only be determined by proteomic
methodologies.methodologies.-- The localization of gene products.The localization of gene products.-- The proteinThe protein--protein interactions.protein interactions.-- Proteins are the most drug targets.Proteins are the most drug targets.
" Proteins, not genes hold the keys to biology "International Business Communication, February 1999
" Proteins: The business end of biology "BioVenture View, April 2000~ Only a protein-based approach will be able
to detect these changes and information ~
4
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) 405:837-846.5
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- Peptide mapping
Bioinformatics 生物資訊
- Protein database- Database mining
Others- Automation (ICAT)- 2D Column chromatography
(MDLC)- Protein chips- BIACORE (biosensor)- Yeast two hybrid system
6
Two-Dimensional Gel Electrophoresis (2DE)proteinprotein solubilizationsolubilization
7AmershamAmersham BiosciencesBiosciences
isoelectricisoelectric focusingfocusing
酸性增強酸性增強 鹼性增強鹼性增強
蛋白質負電荷減弱蛋白質負電荷減弱 蛋白質正電荷減弱蛋白質正電荷減弱
2DE - Sample Preparation
SDSSDS--PAGE by molecular weightPAGE by molecular weight
8
2DE – Detection by Staining Reagent
Silver
Coomassie blue
9
2DE – Detection by Staining Reagent
Silver Coomassie Sypro Ruby
Cy3 Cy3 + Cy5 Cy510
2DE - Differential Display
11
2DE - Image Processing
50% 50% increaseincrease50% 50% decreasedecrease
12
2D Databases on Internethttp://tw.expasy.org
13
Drawbacks of 2DE1. 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 post-translationally modified proteins.
14
Mass SpectrometersQQ--TofTof 2 (2 (MicromassMicromass UK)UK)
M@LDI (M@LDI (MicromassMicromass UK)UK)
ESIESI--MS/MS MS/MS
MALDIMALDI--TOF TOF
15
Principle of Mass Spectrometry質譜儀質譜儀 ((Mass Mass Spectrometers)Spectrometers)
-- 樣品進量樣品進量
-- 樣品離子化樣品離子化
-- 質量偵測質量偵測
-- 離子數目偵測離子數目偵測
質譜質譜 ((Mass Spectrum)Mass Spectrum)
-- molecular weightmolecular weight((分子量)分子量)
-- fragmentation informationfragmentation information((結構)結構)
16
Tandem Mass Spectrometry
17
胺基酸鍵斷裂胺基酸鍵斷裂Collision-induced dissociation (CID)
胺基酸定序胺基酸定序 SequencingSequencing
Tandem MS-based Protein Sequencing
18
471.78
472.29
472.78
•• ConcentrationConcentration--dependent processdependent process•• Favor multiple charged species Favor multiple charged species
Protein Identification - Sequencinghttp://www.matrixscience.com
19
Sequencing Search Result
20
MALDI-TOF Mass Spectrometry
21
MatrixMatrix--Assisted Laser Assisted Laser DesorptionDesorption/Ionization /Ionization TimeTime--ofof--FlightFlight
Protein Identification - Peptide MappingPeptide mass fingerprinting (PMF) Peptide mass fingerprinting (PMF)
*
*
**
**
*
Trypsin
22
Protein Identification - Peptide Mappinghttp://www.matrixscience.com
23
Peptide Mapping Search Result
24
Protein Chip technologyProtein chips Protein chips –– a variety of a variety of ““baitbait’’ proteins as ELISAproteins as ELISA
Depend on specific and well-characterized antibodies and a number of technical problems.
Nature (2000) 405:837-846.25
ICAT technologyQuantitationQuantitation by mass spectrometryby mass spectrometry
-- but using limited or no protein separationbut using limited or no protein separation
Nature biotechnology (1999) 17:994-999.
Quantitative analysis of complex protein mixture using isotope-coded affinity tags (ICAT)
26
Multi-Dimensional technologyIEF-NP RP HPLC
- Rotofor (BioRad)- 1g of total protein
27
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
Protein-Protein InteractionsNature (2002) 415:123-124.
1739 baits, 1440 proteins, 232 complexes
Nature (2002) 415:180-183.
Nature (2002) 415:141-147.
725 baits, 3617 interactions, 1578 proteins
29
BIACORESurface Surface PlasmonPlasmon Resonance BiosensorResonance Biosensor
θ
IncidentL ight
D etector
E vanescentField
-Real time biomolecular interaction analysis
-Non-invasive optical measuring technique-No labeling technique
30
BioEssays (1998) 20:1-6.
Yeast Two-Hybrid SystemMolecular & Cellular Proteome (2002) 1:561-566.
31
WebGenNet(genome.c.kanazawa-u.ac.jp/webgen/webgen.html)
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- Peptide mapping
Bioinformatics 生物資訊
- Protein database- Database mining
Others- Automation (ICAT)- 2D Column chromatography
(MDLC)- Protein chips- BIACORE (biosensor)- Yeast two hybrid system
32
Activity-Based Probe (ABP)
PNAS (1999) 96:14694-14699.33
Properties of Proteomics Techniques
Current Opinion in Chemical Biology (2002) 6:427-433.34
Properties of Proteomics Techniques
Current Opinion in Chemical Biology(2002) 6:427-433.
35
Applications of Proteomics TechnologyEstablishing and mining proteome from different speciesIn 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.
http:/http://tw.expasy/tw.expasy.org/ch2d/2d.org/ch2d/2d--index.htmlindex.html
36
Applications of Proteomics TechnologyDrug Target/marker identificationThis 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.
1.1. Body fluidsBody fluids1.11.1 Blood cellsBlood cells
1.1.11.1.1 ErythrocytesErythrocytes1.1.21.1.2 LeukocytesLeukocytes1.1.31.1.3MonocytesMonocytes/macrophages/macrophages1.1.41.1.4 LymphocytesLymphocytes1.1.51.1.5 PlateletsPlatelets
1.21.2 Plasma and serumPlasma and serum1.31.3 UrineUrine1.41.4 Amniotic fluidAmniotic fluid1.51.5 Cerebrospinal fluidCerebrospinal fluid1.61.6 SynovialSynovial fluidfluid1.71.7 SalivaSaliva1.81.8 SweatSweat1.91.9 TearsTears1.101.10 SemenSemen1.111.11 Other fluids
2.2. Solid tissuesSolid tissues2.12.1 HeartHeart2.22.2 BrainBrain2.32.3 ThyroidThyroid2.42.4 MuscleMuscle
3.3. Malignant Malignant diseasesdiseases
4.4. Tissue cultureTissue culture5.5. Malignant cellsMalignant cells6.6. Bacterial proteinsBacterial proteins
Other fluids 37
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-naphthyl-amine and benzidine) and the parasite Schistosoma haematobium.
- Transitional Cell Carcinoma (TCC)- Squamous Cell Carcinoma (SCC)
SCC
TCC
ElectrophoresisElectrophoresis (1999) 20:349(1999) 20:349--354.354.
Current status:ELISA kit: 1-5 µg/mL (~ 10-7 M)
38
Creutzfeldt-Jakob disease (CJD)CJD)庫茲德賈克氏病庫茲德賈克氏病-- 一種會傳染的神經疾病,具有一種會傳染的神經疾病,具有
和瘋牛病同類的病原和瘋牛病同類的病原,,是一種是一種
變性的蛋白質,喜歡侵襲神經變性的蛋白質,喜歡侵襲神經
系統。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.
HsiechHsiech et al. et al. N N EnglEngl J Med J Med (1996) 335:924(1996) 335:924--930.930. 39
Red: Animals with clinical dilated cardiomyopathy.
Green: Normal animals.Purple:Clinically normal
but are offspring of two parents with clinical dilated cardiomyopathy.
40
Proteomics & drug mode-of-action studiesFluorouracil (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
Measurement of Drug ToxicityThe 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
Measurement of Drug ScreeningStudies 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
SCIENTIFIC AMERICANSCIENTIFIC AMERICAN, APRIL 2002 p41, APRIL 2002 p41
The Impact on Drug Discovery & Therapy
• Identification of drug targets
• Action mechanism of drugs
• Drug toxicity and screening
• Lead optimization