해외전시회 표준참가계획서 작성 매뉴얼 · 대부분 “여기 당신의 여행 일정표와 부스 스케줄이 있어요. 그곳에서 봅시다.”와 같은 정도는
Basic Scheme of Biological Phenomena Signals. Proteome 분석기술의 필요성 생체 내...
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Transcript of Basic Scheme of Biological Phenomena Signals. Proteome 분석기술의 필요성 생체 내...
Basic Scheme of Biological Phenomena
Signals
Proteome 분석기술의 필요성 • 생체 내 기능은 단백질이 한다 ( 약 30,000 종류 ).• 단백질 발현 정도는 mRNA 양과 다르다 . • 단백질은 다양한 modification 이 많다 .
• 이를 분석할 수 있는 high throughput proteome 분석 기술이 필요하다 .
Application of Proteome Analysis
Protein modification
Differential protein expression
Protein-protein interaction
Discovery of target proteins
Development of drug marker
Screening of chemical library
Development of therapeutic and diagnostic drugs
Basic life science Growth, proliferation, aging, death Diseases암 , 면역 / 뇌 / 심혈관질환등Adaptation to environment
Proteome analysis
Sample preparation• Prefractionation
Proteome separation
Spot analysis• peptide fingerprinting • peptide sequencing
Identification of proteins
Functional studies of identified
proteins
MALDI-TOF-MS
Differentially Expressed proteins
Changes in protein-protein interaction
Protein modification
Database
Protein Overexpression, Cell line,mutant construction, antibody production
Development of new drug target
Comparison with Genebank(Bioinformatics)
Spot excisionin gel
digestion
2D - PAGE IEF
SDS
Mass (m/z)
0
2000
4000
6000
8000
10000
Co
un
ts
1000 1500 2000 2500 3000
Proteome Separation : 2-D PAGE(2-Dimensional polyacrylamide gel electrophoresis)
• Organ, Tissue, Cells• Sample Preparation• 2-D gel
Electrophoresis– IEF & Mol. wt
• Stain and/or Blot• Image Analysis
IEF
SD
S-
PA
GE
2D gel electrophoresis
1. Samples prep – denatured and solubilized using urea, a non-ionic detergent, IPG Buffer, and a reducing agent
2. Apply rehydration solution
3. Lay IPG strip
from www.apbiotech.com
4. Apply protein sample
5. Apply oil
6. Place cover
7. Place assembled strip holder on IPGphor platform and run
from www.apbiotech.com
Ettan DALT II System
8. After a precast gel is loaded, the IPG strip is added, sealed into position, and the cassette is placed into the Ettan DALT II Separation Unit
IEF
SD
S-
PA
GE
9. Spot detection & analysis
from www.apbiotech.com
Peptides from Proteins
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
intact protein
enzyme
peptide fragments
Pumping
Laser
Sample plate
Extractiongrids
Pumping
Lineardetector
Timed ionselector
Reflectordetector
Reflector
Disorbedions
Matrix Sample
UV laser337 nm
MALDI-TOF MSMatrix-Assisted Laser Desorption/Ionization Time Of Flight MS
Camera
MALDI-TOF MS
STR (PerSepive)MALDI TOF MSdelayed extractionReflectorHigh current detector STR (PerSepive)
Automatic Data Acquisition of Gel Spot
Database Search Results
Conclusions
Proteomics using 2-D gel electrophoresis and MALDI-TOF MS are valuable analytical tool for identification of
Differentially expressed proteins Protein modifications
Protein-protein interactionPeptide sequencing