Application of Proteomics in Biological Research

1
Application of Proteomics in Biological
Research An introduction
Jau-Song Yu Department of Cell and Molecular
Biology Chang Gung University
2
The central dogma of life science
Gene (DNA) mRNA Protein
Transcription 1000 X Amplification Tra
nslation 100 X Amplification
3
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
4
Human Genome Project (HGP) --- 99 sequence of
human genome published
16 February 2001Volume 291Number 5507The Human
Genome
15 February 2001, Volume 409, Number 6822
5
Global gene expression analysis --- cDNA
microarray
Breast cancer samples vs. normal tissues
PNAS USA 98, 1086910874 (2001)
6
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 stability, alternative splicing,
etc. Post-translational Modification of
proteins (covalent modification, proteolytic
cleavage, activator, inhibitor, etc)
C2H5 PO4
7
Genomics genes characterization and
identification
Proteomics functional analysis of gene
products
Bioimformatics
8
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
9
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.
10
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.
11
General principle and protocol of 2-dimension gel
electrophoresis
sample
Isoelectric focusing (1st dimension)
Ampholytes
pH 9 - pH 3
polyacrylamide
2nd dimension
SDS-PAGE
MW
pH gradient
12
Traditional Equipment for Isoelectric focusing
(IEF)
Ampholytes polyacrylamide
Cathode (-) electrode solution
Anode () electrode solution
13
Immobilized pH Gradient (IPG)
Acidic buffering group
COO-
Acrylamide monomer
CH2 - CH-C-NH-R
Basic buffering group
NH3
O
Polyacrylamide gel
14
Production of Immobilized pH Gradient (IPG) strip
Gradient maker
A
D
plastic support film
B
E
C
F
pH 3 pH 10
15
Equipment for Isoelectric focusing (IEF)
IPGphor (IEF System) Amersham Pharmacia Biotech
Inc.
Protein IEF Cell Bio-Rad Laboratories
16
Sample preparation
Lysis solution 8M Urea 4
NP-40 or CHAPS 40mM Tris base
Cell line
Lysis solution
Sonication
vacuum
Lysis solution
Centrifugation
Measurement of protein
2-DE sample
17
IPG strip rehydration and sample loading
Rehydration solution
2-DE sample
Rehydration solution 8M Urea 2
NP-40 or CHAPS 2 IPG buffer
(Ampholyte) 0.28 DTT Trace
Bromophenol blue
IPG strip holder
Position the IPG strip
18
IPG strip rehydration and sample loading
Strip holder
Anode () electrode
Cathode (-) electrode
30 voltage 12hr
19
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
20

• Second dimension SDS-PAGE
• SDS equilibration
• SDS-PAGE

SDS equilibration buffer 50 mM Tris-HCl 6
M Urea 30 Glycerol 2
SDS Trace Bromophenol
IPG strip
21
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
22
2-DE separation of soluble E. coli proteins
23
For cancer study
Clinical specimens
Cryostat
Laser-captured microdissector (LCM)
Normal cells
Tumor cells
2D gel electrophoresis Immage system
SDS-PAGE
isoelectrofocusing
(?????)
24
Identification of 2-DE-separated proteins in a
high-throughput way using biomass spectrometry
MALDI TOF/TOF MS
LC/MSn
25
What is a mass spectrometer and what does
it do?
Gary Siuzdak (1996) Mass Spectrometry for
Biotechnology, Academic Press
26
Analogy between mass analysis and the
dispersion of light
27
Components of a mass spectrometer
28
MALDI-TOF MS (Matrix-assisted laser
desorption/ionization-Time of flight) (??????????-
???????)
Second detector
Reflector
Target plate
Time of Flight
Target plate
First detector
Laser
M/Z
29
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.
30
Matrix-assisted laser desorption/ionization source
31
Mass Analyzer-Time of Flight (TOF)
Kinetic Energy ½ mv2
v (2KE/m)1/2
m/z
32
Sensitivity of MALDI-TOF MS
10 fg
1347.7 g/mole x 5 x 10 -18 mole 6.74 x 10 15 g
33
How to identify 2-DE-separated proteins by
MALDI-TOF MS? Linking between genomics/bioinformat
ics/proteomics
Clinical specimens
Cryostat
Laser-captured microdissector (LCM)
Normal cells
Tumor cells
2D gel electrophoresis Immage system
SDS-PAGE
isoelectrofocusing
(?????)
34
(?????)
Digested by trypsin (Lys, Arg)
(854, 931, 935, 1021, 1067, 1184, 1386, 1438)
(621, 754, 778, 835, 1204,, 1398, 1476, 1582)
(664, 711, 735, 904, 1079, 1188, 1438)
(602, 755, 974, 1166, 1244, 1374)
(Masses of tryptic peptides are predictable from
gene sequence databases)
MALDI-TOF MS analysis
(M/Z)
(854, 935, 1021, 1067, 1184, 1386, 1438)
(621, 778, 835, 1204,, 1398, 1582)
(735, 904, 1079, 1188, 1438)
(755, 974, 1244, 1374)
Database search/mapping
Protein identified (100?)
35
An example Identification of specific proteins
purified from pig brain
(A)
(B)
pH
3
10
c
170
b
116.3
2
1
3
a
66.3
1
55.4
29
21.5
36
MALDI-TOF analysis of tryptic fingerprint from
the proteins purified from pig brain
(a1)
(b1)
(b3)
(d2)
(c2)
37
Data base search for the purified protein from
pig brain
(c2)
38
Collapsin Response Mediator Protein-2 (CRMP-2,
human)
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 pI5.95
39
Proteomics solution
IEF
SDS-PAGE
40
Direct identification of the amino acid sequence
of peptides by tandem mass spectrometry
41
Amino acid sequence analysis by MS - an example
2169
908
42
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 2002for 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
macromoleculesand the other half toKurt
WüthrichSwiss Federal Institute of Technology
(ETH), Zürich, Switzerland and The Scripps
Research Institute, La Jolla, USAfor his
development of nuclear magnetic resonance
spectroscopy for determining the
three-dimensional structure of biological
macromolecules in solution.Revolutionary
analytical methods for biomolecules This
years 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.
43
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 years
Nobel Prize award.
44
Brukers movie for MALDI-TOF Mass Spectrometry
45
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