Transcript Proteomics

Application of Proteomics in Biological Research
An introduction
Jau-Song Yu
Department of Cell and Molecular Biology
Chang Gung University
The central dogma of life science
Gene (DNA)
Transcription
1000 X Amplification
mRNA
Translation
100 X Amplification
Protein
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
Human Genome Project (HGP) ---
99% sequence of human genome published
16 February 2001
Volume 291
Number 5507
The Human Genome
15 February 2001, Volume 409, Number 6822
Global gene expression analysis --- cDNA microarray
Breast cancer
samples vs.
normal tissues
PNAS USA 98, 10869–10874 (2001)
The extent of gene expression (i.e. the amount of mRNA) is only
one of the many factors determining the protein function in cells
mRNA level  expressed protein level nor does it indicate
the nature of the functional protein product
mRNA stability,
alternative splicing, etc.
t-RNA
t-RNA
mRNA
Ribosome
t-RNA
(....)
Protein
DNA
t-RNA
Post Translational
Modifications
X
(....)
X
Active Protein
CHO
C
2H5
PO44
PO
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
Post-translational
Modification of proteins
(covalent modification,
proteolytic cleavage,
activator, inhibitor, etc)
Genomics
Proteomics
genes characterization
and identification
functional analysis
of gene products
Bioimformatics
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
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.
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.
NH3+
NH3+
NH2
COOH
COO-
COO-
NH3+
NH3+
NH2
COOH
COO-
COO-
pH < pI
Net positive charge
pH = pI
pH > pI
Net negative charge
3
Isoelectric point
Net charge
2
1
0
-1
-2
-3
2
3
4
5
6
7
pH
8
9
10
11
General principle and protocol of 2-dimension gel electrophoresis
Ampholytes
sample
pH 9
-
pH 3
+
Isoelectric focusing
(1st dimension)
polyacrylamide
MW
2nd dimension
SDS-PAGE
pH gradient
Traditional Equipment for Isoelectric focusing (IEF):
Ampholytes
polyacrylamide
Cathode (-)
electrode
solution
Anode (+)
electrode
solution
Immobilized pH Gradient (IPG)
Acrylamide monomer
Acidic buffering group: COO-
CH2 - CH-C-NH-R
Basic buffering group:
NH3+
O
Polyacrylamide gel
Production of Immobilized pH Gradient (IPG) strip
basic
A
acidic
Gradient maker
plastic support
film
D
B
E
C
F
pH 3
pH 10
Equipment for Isoelectric focusing (IEF):
IPGphor (IEF System)
Protein IEF Cell
Amersham Pharmacia Biotech Inc.
Bio-Rad Laboratories
Sample preparation
Lysis solution:
8M
4%
40mM
Urea
NP-40 or CHAPS
Tris base
Cell line
Lysis solution
Sonication
vacuum
Lysis solution
Centrifugation
Measurement of [protein]
2-DE sample
IPG strip rehydration and sample loading
2-DE sample
Rehydration
solution
Rehydration solution:
8M
2%
2%
0.28%
Trace
IPG strip holder
Position the
IPG strip
Urea
NP-40 or CHAPS
IPG buffer (Ampholyte)
DTT
Bromophenol blue
IPG strip rehydration and sample loading
Strip holder
Anode (+)
electrode
Cathode (-)
electrode
30 voltage 12hr
First dimension: Isoelectric focusing
Electrode
pads
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
Holder cover
IPG strip
Electrode
Voltage
5. 8000 V gradient (?)
Time
36000vhr
Second dimension: SDS-PAGE
• SDS equilibration
• SDS-PAGE
IPG strip
SDS
SDS equilibration buffer
50 mM
6M
30%
2%
Trace
Marker in paper
SDS-PAGE
SDS-PAGE
Tris-HCl
Urea
Glycerol
SDS
Bromophenol
0.5% agarose
in running buffer
SDS-PAGE
Detection of proteins separated on gels --Protocol of silver stain:
50% methanol
25% acetic acid
4hr
ddH2O
30 sec
ddH2O x 3 times
30min/time
3% Na2CO3
0.0185% formaldehyde
0.004% DTT solution
30min
2.3M citric acid
0.1% AgNO3
30min
5% acetic acid
25% methanol
2-DE separation of soluble E. coli proteins
For cancer study ~
Clinical specimens
Cryostat
Laser-captured microdissector (LCM)
Tumor
cells
SDS-PAGE
Normal
cells
2D gel
electrophoresis
Immage system
isoelectrofocusing
(?????)
Identification of 2-DE-separated proteins in a high-throughput
way using biomass spectrometry
MALDI TOF/TOF MS
LC/MSn
What is a mass spectrometer
and what does it do?
Gary Siuzdak (1996) Mass Spectrometry for Biotechnology, Academic Press
Analogy between mass analysis
and the dispersion of light
Components of a mass spectrometer
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 plate
Second detector
Reflector
Reflector
Second Detector
Time of Flight
Target plate
First
detector
First
Detector
Laser
Laser
圖十一：在 MALDI-TOF MS中的反射器設計
M/Z
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.
Matrix-assisted laser desorption/ionization source
Mass Analyzer-Time of Flight (TOF)
Kinetic Energy
= ½ mv2
v = (2KE/m)1/2
m/z
Sensitivity of MALDI-TOF MS
~10 fg
1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g
How to identify 2-DE-separated proteins by MALDI-TOF MS?
Linking between genomics/bioinformatics/proteomics
Clinical specimens
Cryostat
Laser-captured microdissector (LCM)
Tumor
cells
SDS-PAGE
Normal
cells
2D gel
electrophoresis
Immage system
isoelectrofocusing
(?????)
(?????)
Digested by trypsin (Lys, Arg)
(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)
MALDI-TOF MS analysis
(621, 778, 835,
1204,, 1398, 1582)
(735, 904, 1079,
1188, 1438)
(755, 974,
1244, 1374)
(854, 935, 1021,
1067, 1184, 1386, 1438)
Database search/mapping
Protein identified (100%?)
(M/Z)
An example ~ Identification of specific proteins purified from pig brain
(A)
(B)
10
pH
3
c
170
b
116.3
66.3
55.4
29
21.5
2
1
3
1
a
MALDI-TOF analysis of tryptic fingerprint from the proteins purified
from pig brain
(a1)
(b1)
(b3)
(d2)
(c2)
Data base search for the purified protein from pig brain
(c2)
Collapsin Response Mediator Protein-2 (CRMP-2, human)
MSYQGKKNIP
VPGGVKTIEA
TTMIIDHVVP
EMEALVKDHG
NGDIIAEEQQ
YITKVMSKSS
FVTSPPLSPD
IPEGTNGTEE
IAVGSDADLV
KIVLEDGTLH
GPVCEVSVTP
PRRTTQRIVA
RITSDRLLIK
HSRMVIPGGI
EPGTSLLAAF
VNSFLVYMAF
RILDLGITGP
AEVIAQARKK
PTTPDFLNSL
RMSVIWDKAV
IWDPDSVKTI
VTEGSGRYIP
KTVTPASSAK
PPGGRANITS
*908.4 da --- 391-397
GGKIVNDDQS
DVHTRFQMPD
DQWREWADSK
KDRFQLTDCQ
EGHVLSRPEE
GTVVYGEPIT
LSCGDLQVTG
VTGKMDENQF
SAKTHNSSLE
RKPFPDFVYK
TSPAKQQAPP
LG
FYADIYMEDG
QGMTSADDFF
SCCDYSLHVD
IYEVLSVIRD
VEAEAVNRAI
ASLGTDGSHY
SAHCTFNTAQ
VAVTSTNAAK
YNIFEGMECR
RIKARSRLAE
VRNLHQSGFS
*2169.1da --- 533-552
*pI~5.95
LIKQIGENLI
QGTKAALAGG
ISEWHKGIQE
IGAIAQVHAE
TIANQTNCPL
WSKNWAKAAA
KAVGKDNFTL
VFNLYPRKGR
GSPLVVISQG
LRGVPRGLYD
LSGAQIDDNI
Proteomics solution
IEF
SDS-PAGE
Direct identification of the amino acid sequence of peptides by
tandem mass spectrometry
Amino acid sequence analysis by MS - an example
2169
908
Press Release: The Nobel Prize in Chemistry 2002
9 October 2002
The 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. Fenn
Virginia Commonwealth University, Richmond, USA, and
Koichi Tanaka
Shimadzu Corp., Kyoto, Japan
”for their development of soft desorption ionisation methods for mass spectrometric analyses of biological
macromolecules”
and the other half to
Kurt Wüthrich
Swiss 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 threedimensional 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.
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.
Bruker’s movie for MALDI-TOF Mass Spectrometry
長庚大學蛋白質體核心實驗室簡介