蛋白質體學 阮雪芬 jul 18 & 25, 2003. outline the characters of proteins differences...
Post on 15-Jan-2016
251 views
TRANSCRIPT
蛋白質體學
阮雪芬Jul 18 & 25, 2003
Outline The characters of proteins
Differences between protein chemistry & proteomics
Why to study proteome
Proteomics Introduction to proteomics Definitions of proteomics The major techniques in current proteomics Protein-protein interactions
The characters of proteins
DNA 和蛋白質合成的地方
Three Developments Formed the Foundation of the New Biology
The growth of gene, expressed sequence tag (EST), and protein-sequence databases during the 1990s.
The introduction of user-friendly, browser-based bioinformatics tools.
The development of oligonucleotide microarray.
Why to study proteome ?
Why the Transcriptomic Analyses May Not Have Revealed All Proteins ?
Lack of correlation between transcript and disease-associated protein levels
Translocation of a protein in the disease state rather than simply differential levels of the transcript
Novel/uncharacterized genes that are not highly represented within the "closed system" of a cDNA array
Individual proteins
Complete sequence analysis
Emphasis on structure and function
Structural biology
Complex mixtures Partial sequence
analysis Emphasis on
identification by database matching
Systems biology
Protein chemistry Proteomics
Introduction To Proteomics
DNA
mRNA
Proteins
Cell functions
Genome “Genomic
s”
Proteome“Proteomics”
Genomics vs. Proteomics
Generalized Proteomics Scheme
Yarmush & Jayaraman, 2002
Definitions of Proteomics
Definitions of Proteomics First coined in 1995 Be defined as the large-scale
characterization of the entire protein complement of a cell line, tissue, or organism.
Goal: -To obtain a more global and integrated
view of biology by studying all the proteins of a cell rather than each one individually.
The classical definition• Two-dimensional gels of cell lysate and annotation• Two-dimensional gels to visualize differential protein expressionIn the post-genomics era• Protein Identification • Post-translational modifications • Determining Function • Molecular Medicine • Differential display by two-dimensional gels • Protein-Protein Interactions
Definitions of Proteomics
Proteomics Origins
In 1975, the introduction of the 2D gel by O’Farrell who began mapping proteins from E. coli.
The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradation picomole
MS technology has replaced Edman degradation to identify proteins femtomole
How Proteomics Can Help Drug Development
http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18
Why is Proteomics Necessary?
Having complete sequences of genome is not sufficient to elucidate biological function.
A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival
Modifications of proteins can be determined only by proteomic methodologies
It is necessary to determine the protein expression level
The localization of gene products can be determined experimentally
Protein-protein interactions Proteins are direct drug targets.
Jürgen Drews, 2000
Amgen( Applied Molecular Genetics)成立日期: 1980 年 4 月 8 日CEO : Kevin W. Sharer員工人數: 6342市場總值: 698.4 億美元產品項目:重組蛋白藥物 EPOGENR (Epoetin alfa) NEUPOGENR (Filgrastim) INFERGENR (Interferon alfacon-1)
資料來源:彭博資訊社、 Zacks.com , 6/14/2001
各項產品營業收入
資料來源: Amgen, Inc.
The Major Techniques in Current Proteomics
The Major Techniques in Current Proteomics
Two-dimensional electrophoresis IEF strip separation SDS-PAGE gel separation
Mass Spectrometry Protein sequencing Peptide mapping
Others ICAT Yeast two hybrid assay Protein chips
Two-dimensional Gel Approach
Nature 2000, 405, 837-846
3.5 1010
42
6070
150
kDa
pH
Increase of 50%
Decrease of 50%
Unmatched spots
Matched spots
Image Matching
www.expasy.ch/ch2d
http://www.expasy.ch/melanie/
Standard Proteome Analysis by 2DE-MS
Current Opinion in Chemical Biology 2000, 4:489–494
Mass Fingerprint Searching in http://www.expasych/tools/peptident.html
Yarmush & Jayaraman, 2002
Typical mass spectrometry scheme
peptide mass mapping and tandem mass spectrometry
Ionization State as a Function of pH
First dimension: IEF (based on isoelectric point)
SD
S-P
AG
E(based on m
olecular w
eight)
+ -
acidic basic
HighMW
Low MW
Sample
Two-dimensional Gel Electrophoresis
Silver staining Coomassie blue staining Sypro Ruby staining
Staining of Polyacrylamide Gels
Image Analysis
In-gel Digestion Enzyme:
trypsin chymotrypsin
*
*
**
**
*
Trypsin
Peptide mass fingerprinting (PMF) or peptide mapping
Mass Spectrometric Identification of Proteins Mapping
1. Cut protein spot 2. Protein digestion
3. Peptide purification4. Spot onto MALDI chip
5. MALDI-TOF analysis 6. Peptide fragment fingerprint
Protease
Protein Identification by MALDI-TOF
IonizationIonization
Sample Sample inputinput AnalyzerAnalyzer DetectorDetector
How Does a Mass Spectrometer Work?
• Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc.
• Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization
(MALDI) etc
• Analysis: quadrupole, time of flight(TOF), ion trap etc.
• Detection:
How Does a Mass Spectrometer Work?
Electrospray
Ionization
Matrix-Assisted Laser Desorption/Ionization (MALDI)
Ionization
Matrix:Matrix:
- organic acids- organic acids
- benzoic acids- benzoic acids
Isotope-coded Affinity Tags (ICAT)
Avidin chromatography
Biotin
Linker
Thiol-reactive end group
ICAT consists of a biotin affinity group, a linker region that can incorporate heavy or light atoms , and a thiol-reactive end group for linkage to cysteines
NATURE, VOL 405, 15 JUNE 2000
A Strategy for Mass Spectrometric Identification of Proteins and Post-translational Modifications
‘proteome chip’ composed of 6,566 protein samples representing 5,800 unique proteins, which are spotted in duplicate on a single nickelcoated glass microscope slide39. The immobilized GST fusion proteins were detected using a labeled antibody against GST.
(MacBeath G. Nat Genet 2002 Dec;32 Suppl 2:526-32 )
Proteome chip
Microarrays for Genomics and Proteomics
DNA microarray are used for genetic analysis as well as expression analysis at the mRNA level.
Protein microarrays are used for expression analysis at the protein level and in the expansive field of interaction analysis.
Protein Microarrays In Medical Research Accelerate immune diagnostics. The reduction of sample volume----the anal
ysis of multiple tumor markers from a minimun amount of biopsy material.
New possibilities for patient monitoring during disease treatment and therapy will be develpoed based on this emerging technology.
Clinical and Biomedical Applications of Proteomics
An approach complementary to genomics is required in clinical situations to better understand epigenetic regulation and get closer to a "holisitic" medical approach.
The potential clinical applications of 2-D PAGE, especially to the analysis of body fluids and tissue biopsies.
Identifying the origin of body fluid samples or the origin of a tissue biopsy.
Analyzing protein phenotypes and protein post-translational modifications in fluid, cells, or tissues.
Examining the clonality of immunoglobulins and detecting clones which are not seen with conventional techniques.
Monitoring disease processes and protein expression. Discovering new disease markers and/or patterns in body
fluids, cells, or tissues.
Clinical applications of 2-D electrophoresis Body fluids
Blood cell Plasma and serum Urine Cerebrospinal fluid Amniotic fluid Synovial fluid Saliva Sweat Tears Semen
Solid tissue Heart Brain Thyroid Muscle
Malignant diseases Tissue culture Malignant cells Bacterial proteins
Young & Tracy Journal of Chromatography A, 698 (1995) 163-179
Protein-protein Interactions
Protein-protein Interactions Introduction Mass Spectrometry Yeast Two-hybrid Assay
Introduction Protein-protein interactions are
intrinsic to every cellular process.
Form the basis of phenomena DNA replication and transcription Metabolism Signal transduction Cell cycle control Secretion
The Study of Protein-protein Interactions by Mass Spectrometry
bait
S14
??
??
* *
**
SDS- PAGE
MASS
Yeast Two-hybrid System Useful in the study of various interactions The technology was originally developed
during the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature).
Yeast Two-hybrid System
GAL4 DNA-binding
domain
GAL4 DNA-activation domain
Nature, 2000
Yeast Two-hybrid System
Nature, 2000
Library-based yeast two-hybrid screening method
Protein-protein Interactions
on the Web Yeast http://depts.washington.edu/sfields/yplm/data/index.html http://portal.curagen.com http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://www.pnas.org/cgi/content/full/97/3/1143/DC1 http://dip.doe-mbi.ucla.edu/ http://genome.c.kanazawa-u.ac.jp/Y2H C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html
Pathway SoftwareBIOCARTA
http://biocarta.com/
Browse all pathway
Pathway SoftwareBIOCARTA
Pathway Result 1:Enolase Glycolysis
Pyruvate
Acetyl-CoAethanol lactate
Cancer cells
BIOCARTA
Pathway Result 2:Retinoic Acid Receptor RXR-alpha
BIOCARTA
Useful BioWebSite name URL Information available
MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse
Peptide mass mapping and sequencing
ProFound http://prowl.rockefeller.edu/cgi-bin/ProFound
Peptide mass mapping and sequencing
PeptIdent http://www.expasy.ch/tools/peptident. Peptide mass mapping and sequencing
PepSea http://195.41.108.38/PepSeaIntro.html
Peptide mass mapping and sequencing
MASCOT http://www.matrixscience.com/ Peptide mass mapping and sequencing
PepFrag http://www.proteometrics.com/ Peptide mass mapping and sequencing
Protein Prospector
http://prospector.ucsf.edu/ Peptide mass mapping and sequencing
FindMod http://www.expasy.ch/tools/findmod/ Posttranslational modification
SEAQUEST http://fields.scripps.edu/sequest/ Uninterpreted MS/MS searchingFASTA Search Programs
http://fasta.bioch.virginia.edu/ Protein and nucleotide database searching
Cleaved Radioactivity ofPhosphopeptides
http://fasta.bioch.virginia.edu/crp Protein phosphorylation site mapping
Major Directions in Coming Proteomics
Chemical proteomics (screens for activity and binding)
Structural proteomics (target validation and development)
Interaction proteomics (identification of new protein targets)
Bioinformatics (annotation of the proteome)
Major Directions in Coming Proteomics
Protein structure prediction and modeling
Assignment of protein structure to genomes
Classifications of protein structures Drug discovery and development
Types of Proteomics and Their Applications to Biology
Proteomics Network
Identify Proteins
Drug Discovery
Structures
Protein-Protein Interactions
Pathways
Protein Functions
Thank You!