application of proteomics in biological research an introduction jau-song yu department of cell and...
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Application of Proteomics in Biological ResearchAn introduction
Jau-Song Yu
Department of Cell and Molecular BiologyChang Gung University
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The central dogma of life science
Transcription 1000 X Amplification
Translation 100 X Amplification
Gene (DNA)
mRNA
Protein
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Genomics:
---Identification and characterization of genes (gene expression) and their arrangement in chromosomes
Proteomics (Functional Genomics):
---Functional analysis of gene products (proteins)
Bioinformatics:
---Storage, analysis and manipulation of the information from genomics and proteomics
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Human Genome Project (HGP) --- 99% sequence of human genome published
16 February 2001Volume 291Number 5507
The Human Genome
15 February 2001, Volume 409, Number 6822
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PNAS USA 98, 10869–10874 (2001)
Global gene expression analysis --- cDNA microarray
Breast cancersamples vs.normal tissues
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The extent of gene expression (i.e. the amount of mRNA) is only one of the many factors determining the protein function in cells
DNA
mRNA
t-RNA
t-RNA
t-RNA t-RNA
Ribosome
(....)
Protein
CHOPO4
(....)
Post TranslationalModifications
X
X
Active Protein
mRNAlevel expressed protein level nor does it indicate the nature of the functional protein product
圖一:DNA序列是藍圖決定細胞的表現,蛋白質卻是實際上有功能的工作者;分子階層的蛋白質及DNA分析是瞭解其功能的關鍵
C2H5
PO4
mRNA stability,alternative splicing, etc.
Post-translationalModification of proteins(covalent modification,proteolytic cleavage, activator, inhibitor, etc)
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Genomics genes characterization and identification
Proteomics functional analysis of gene products
Bioimformatics
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Proteomics ---
Global analysis of hundreds to thousands of proteins in cells or tissues simultaneously (why we need?)
How to analyze hundreds to thousands of proteins in cells or tissues simultaneously?
● Separation of proteins on one matrix --- two-dimensional gel electrophoresis ● Identification of separated proteins in a high-throughput way --- biomass spectrometry
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2-Dimension Electrophoresis (2-DE) for Protein Separation
One of the core technology of
proteomics is 2-DE: At present, there is
no other technique which is capable of
resolving thousands of proteins in one
separation procedure.
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Isoelectric point (pI): Isoelectric point is the pH of a solution at which the net charge of protein is
zero. In electrophoresis there is no motion of the particles in an electric
field at the isoelectric point.
Net
cha
rge
-3
-2
-1
0
1
2
3
2 3 4 5 6 7 8 9 10 11
pH
Isoelectric point
NH3+
COOH
NH3+
COOH
pH < pINet positive charge
NH3+
COO-
NH3+
COO-
pH = pI
NH2
COO-
NH2
COO-
pH > pINet negative charge
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sample
pH 9 -
pH 3 +
Isoelectric focusing(1st dimension)
General principle and protocol of 2-dimension gel electrophoresis
MW
pH gradient
SDS-PAGE
Ampholytes
polyacrylamide
2nd dimension
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Traditional Equipment for Isoelectric focusing (IEF):
Ampholytespolyacrylamide
Cathode (-) electrode solution
Anode (+) electrode solution
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Immobilized pH Gradient (IPG)
Polyacrylamide gel
Acidic buffering group:
Basic buffering group:CH2 - CH-C-NH-R
O
COO-
NH3+
Acrylamide monomer
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Gradient maker
plastic support film
Production of Immobilized pH Gradient (IPG) strip
A
C
B
F
E
Dacid
ic
basi
c
pH 3
pH 10
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IPGphor (IEF System)Amersham Pharmacia Biotech Inc.
Protein IEF CellBio-Rad Laboratories
Equipment for Isoelectric focusing (IEF):
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Lysis solution:8M Urea4% NP-40 or CHAPS40mM Tris base
Sample preparation
Cell line
Lysis solution
Sonication
vacuum
Lysis solution
Centrifugation
Measurement of [protein]
2-DE sample
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IPG strip rehydration and sample loading
2-DE sample Rehydration solution
Rehydration solution:8M Urea2% NP-40 or CHAPS2% IPG buffer (Ampholyte)0.28% DTTTrace Bromophenol blue
IPG strip holder
Position the
IPG strip
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IPG strip rehydration and sample loading
Strip holder
Cathode (-) electrode
Anode (+) electrode
30 voltage 12hr
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First dimension: Isoelectric focusing
1. Place electrode pads (?)
2. 200 V step-n-hold 1.5hr
3. 500 V step-n-hold 1.5hr
4. 1000 V gradient 1500vhr
5. 8000 V gradient (?) 36000vhr
Time
Vol
tage
Holder cover
IPG strip
Electrode
Electrode pads
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Second dimension: SDS-PAGE• SDS equilibration• SDS-PAGE
SDS equilibration buffer50 mM Tris-HCl6 M Urea30% Glycerol2% SDSTrace Bromophenol
SD
S
SDS-PAGE SDS-PAGE
0.5% agarose in running buffer
SDS-PAGE
Marker in paper
IPG strip
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Detection of proteins separated on gels ---Protocol of silver stain:
50% methanol25% acetic acid4hr
ddH2O x 3 times
30min/time
0.004% DTT solution30min
0.1% AgNO3
30min
ddH2O
30 sec
3% Na2CO3
0.0185% formaldehyde
2.3M citric acid
5% acetic acid25% methanol
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2-DE separation of soluble E. coli proteins
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For cancer study ~
Normal cells
Tumor cells
SD
S-P
AG
E
isoelectrofocusing
Laser-captured microdissector (LCM)
(?????)
Clinical specimens Cryostat
2D gel electrophoresis
Immage system
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Identification of 2-DE-separated proteins in a high-throughput way using biomass spectrometry
MALDI TOF/TOF MS LC/MSn
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What is a mass spectrometer and what does it do?
Gary Siuzdak (1996) Mass Spectrometry for Biotechnology, Academic Press
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Analogy between mass analysis and the dispersion of light
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Components of a mass spectrometer
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MALDI-TOF MS (Matrix-assisted laser desorption/ionization-Time of flight)(基質輔助雷射脫附游離 -飛行時間質譜儀 )
Target plateFirst DetectorLaser
Reflector
Second Detector
圖十一:在 MALDI-TOF MS中的反射器設計
0
40000
40 80 120 160
RPKPQQFFGLMamide
m/z
0
40000
40 80 120 160
RPKPQQFFGLMamide
m/z
M/Z
Time of Flight
Laser
First detector
Second detector ReflectorTarget plate
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MALDI matrix
# A nonvolatile solid material that absorbs the laser radiation resulting in the vaporization of the matrix and sample embedded in the matrix.
#The matrix also serves to minimize sample damage from the laser radiation by absorbing most of the incident energy and the matrix is believed to facilitate the ionization process.
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Matrix-assisted laser desorption/ionization source
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Mass Analyzer-Time of Flight (TOF)
Kinetic Energy = ½ mv2
v = (2KE/m)1/2
m/z
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Sensitivity of MALDI-TOF MS
~10 fg
1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g
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How to identify 2-DE-separated proteins by MALDI-TOF MS?Linking between genomics/bioinformatics/proteomics
Normal cells
Tumor cells
SD
S-P
AG
E
isoelectrofocusing
Laser-captured microdissector (LCM)
(?????)
Clinical specimens Cryostat
2D gel electrophoresis
Immage system
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(?????)
MALDI-TOF MS analysis
Digested by trypsin (Lys, Arg)
Database search/mapping
Protein identified (100%?)
(621, 754, 778, 835,1204,, 1398, 1476, 1582)
(664, 711, 735, 904,1079, 1188, 1438)
(602, 755, 974,1166, 1244, 1374)
(854, 931, 935, 1021,1067, 1184, 1386, 1438)
(Masses of tryptic peptides are predictable from gene sequence databases)
(621, 778, 835,1204,, 1398, 1582)
(735, 904, 1079, 1188, 1438)
(755, 974, 1244, 1374)
(854, 935, 1021,1067, 1184, 1386, 1438) (M/Z)
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170
116.3
66.3
55.4
29
21.5
pH 310
a
bc
(B)
1
2
1 3
An example ~ Identification of specific proteins purified from pig brain
(A)
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(d2)
(a1)
(b1)
(b3)
MALDI-TOF analysis of tryptic fingerprint from the proteins purified from pig brain
(c2)
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Data base search for the purified protein from pig brain
(c2)
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MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEA HSRMVIPGGI DVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRD IGAIAQVHAE NGDIIAEEQQ RILDLGITGP EGHVLSRPEE VEAEAVNRAI TIANQTNCPL YITKVMSKSS AEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTL IPEGTNGTEE RMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLV IWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLH VTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPP VRNLHQSGFS LSGAQIDDNI PRRTTQRIVA PPGGRANITS LG
*908.4 da --- 391-397 *2169.1da --- 533-552 *pI~5.95
Collapsin Response Mediator Protein-2 (CRMP-2, human)
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Proteomics solution
IEF
SD
S-P
AG
E
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Direct identification of the amino acid sequence of peptides bytandem mass spectrometry
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Amino acid sequence analysis by MS - an example
2169
908
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Press Release: The Nobel Prize in Chemistry 2002 9 October 2002The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2002”for the development of methods for identification and structure analyses of biological macromolecules” with one half jointly to John B. FennVirginia Commonwealth University, Richmond, USA, andKoichi TanakaShimadzu Corp., Kyoto, Japan ”for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules”
and the other half toKurt WüthrichSwiss Federal Institute of Technology (ETH), Zürich, Switzerland and The Scripps Research Institute, La Jolla, USA”for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution”.
Revolutionary analytical methods for biomolecules This year’s Nobel Prize in Chemistry concerns powerful analytical methods for studying biological macromolecules, for example proteins. The possibility of analysing proteins in detail has led to increased understanding of the processes of life. Researchers can now rapidly and simply reveal what different proteins a sample contains. They can also determine three-dimensional pictures showing what protein molecules look like in solution and can then understand their function in the cell. The methods have revolutionised the development of new pharmaceuticals. Promising applications are also being reported in other areas, for example foodstuff control and early diagnosis of breast cancer and prostate cancer. Mass spectrometry is a very important analytical method used in practically all chemistry laboratories the world over. Previously only fairly small molecules could be identified, but John B. Fenn and Koichi Tanaka have developed methods that make it possible to analyse biological macromolecules as well. In the method that John B. Fenn published in 1988, electrospray ionisation (ESI), charged droplets of protein solution are produced which shrink as the water evaporates. Eventually freely hovering protein ions remain. Their masses may be determined by setting them in motion and measuring their time of flight over a known distance. At the same time Koichi Tanaka introduced a different technique for causing the proteins to hover freely, soft laser desorption. A laserpulse hits the sample, which is “blasted” into small bits so that the molecules are released. The other half of the Prize rewards the further development of another favourite method among chemists, nuclear magnetic resonance, NMR. NMR gives information on the three-dimensional structure and dynamics of the molecules. Through his work at the beginning of the 1980s Kurt Wüthrich has made it possible to use NMR on proteins. He developed a general method of systematically assigning certain fixed points in the protein molecule, and also a principle for determining the distances between these. Using the distances, he was able to calculate the three-dimensional structure of the protein. The advantage of NMR is that proteins can be studied in solution, i.e. an environment similar to that in the living cell.
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The Nobel Prize in Chemistry for 2002 is to be shared between scientists working on two very important methods of chemical analysis applied to biological macromolecules: mass spectrometry (MS) and nuclear magnetic resonance (NMR). Laureates John B. Fenn, Koichi Tanaka (MS) and Kurt Wuthrich (NMR) have pioneered the successful application of their techniques to biological macromolecules. Biological macromolecules are the main actors in the makeup of life whether expressed in prospering diversity or in threatening disease. To understand biology and medicine at molecular level where the identity, functional characteristics, structural architecture and specific interactions of biomolecules are the basis of life, we need to visualize the activity and interplay of large macromolecules such as proteins. To study, or analyse, the protein molecules, principles for their separation and determination of their individual characteristics had to be developed. Two of the most important chemical techniques used today for the analysis of biomolecules are mass spectrometry (MS) and nuclear magnetic resonance (NMR), the subjects of this year’s Nobel Prize award.
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Bruker’s movie for MALDI-TOF Mass Spectrometry
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長庚大學蛋白質體核心實驗室簡介