Application of Proteomics in Biological ResearchAn introduction

Jau-Song Yu

Department of Cell and Molecular BiologyChang Gung University

The central dogma of life science

Transcription 1000 X Amplification

Translation 100 X Amplification

Gene (DNA)

mRNA

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 2001Volume 291Number 5507

The Human Genome

15 February 2001, Volume 409, Number 6822

PNAS USA 98, 10869–10874 (2001)

Global gene expression analysis --- cDNA microarray

Breast cancersamples vs.normal tissues

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)

Genomics genes characterization and identification

Proteomics 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.

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

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

Traditional Equipment for Isoelectric focusing (IEF):

Ampholytespolyacrylamide

Cathode (-) electrode solution

Anode (+) electrode solution

Immobilized pH Gradient (IPG)

Polyacrylamide gel

Acidic buffering group:

Basic buffering group:CH2 - CH-C-NH-R

O

COO-

NH3+

Acrylamide monomer

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

IPGphor (IEF System)Amersham Pharmacia Biotech Inc.

Protein IEF CellBio-Rad Laboratories

Equipment for Isoelectric focusing (IEF):

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

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

IPG strip rehydration and sample loading

Strip holder

Cathode (-) electrode

Anode (+) electrode

30 voltage 12hr

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

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

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

2-DE separation of soluble E. coli proteins

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

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 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

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

Normal cells

Tumor cells

SD

S-P

AG

E

isoelectrofocusing

Laser-captured microdissector (LCM)

(?????)

Clinical specimens Cryostat

2D gel electrophoresis

Immage system

(?????)

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)

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)

(d2)

(a1)

(b1)

(b3)

MALDI-TOF analysis of tryptic fingerprint from the proteins purified from pig brain

(c2)

Data base search for the purified protein from pig brain

(c2)

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)

Proteomics solution

IEF

SD

S-P

AG

E

Direct identification of the amino acid sequence of peptides bytandem mass spectrometry

Amino acid sequence analysis by MS - an example

2169

908

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.

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