8 蛋白質科技 Protein Technology

8.1 分子量測定 Molecular weight

有許多方法可以測定蛋白質分子量

8.2 蛋白質構造與組成 Structure analysis

蛋白質構造與組成的細部分析

8.3 免疫學工具 Immunological tools

抗体也是檢定蛋白質的重要工具

8.4 蛋白質新世界 Protein new world

探討蛋白質的新觀念與新科技不斷浮現

1

Mass

質譜儀

Proteomics

8.1 分子量決定法 Molecular weight determination

8.1.1 膠体過濾法 Gel filtration

依蛋白質分子量與分子体積的大小測定

8.1.2 梯度電泳法 Gradient PAGE

可佐證原態分子量之測定

8.1.3 其它分子量測定方法 Other methods

超高速離心法 Ultracentrifugation

由胺基酸序列計算 Deduced from amino acid sequence

質譜儀分析 Mass spectrometry

2

Mass 質譜儀

參考 2.2

參考 3.2

8.1.1 膠体過濾法依原態分子量分離 Native MW

XY

Z

EEnzyme

activity

Elution Volume (mL)

A

VoVe

Vt0

3

(1) 分子量與 Elution volume (Ve) 成反比

(2) 與已知分子量之標準品比較

AF IEX GF TP XT marker AF IEX GF TP XT marker

252

kD

98

64

50

36

GUS

252

kD

98

64

50

36

GUS

10% SDS-PAGE Western Transfer

← 純化步驟順序 Purification steps

MW = 70 kD

■ 以電泳及抗体檢定表現蛋白質 GUS

Expressed protein GUS is detected by SDS-PAGE and immunostaining

4

MW

5

6

4

10

10

3

10

10

MW=260 kD

GUS

1

2

3

0

Sephacryl S-300

1.6 x 90 cmBuffer A-150

a typical result260 kD

70 kD = 4

Vitamin B12

(detected by color)Elution Volume

+

■ 以膠体過濾法求得 GUS 原態分子量

Native molecular weight of GUS is determined by gel filtration

5

Standard curve

Tetramer

8.1.2 製備梯度電泳片 Prepare the gradient gel

5% 20%Upper-limiting

solution

由下方開始

注入梯度溶液Start the gradient

from the bottom

可觀察所拉梯度是否均勻The blue color shows how the

gradient formed in the gel

%5

20

● Determine MW by native-PAGE

(1) Sample protein pI < 8.0

(2) Use gradient gel

(3) Longer running time

Gradient mixer

若在高限溶液中

加入少量染劑Blue dye added in the upper limiting

solution

You can use commercial pre-cast gradient gel (various gel percentage)

6

■ 原態梯度電泳定分子量 Gradient disc-PAGE

kD

669

440

232

140

kD

900800700600

500

400

300

200

100

Migration (cm)

1 2 3 4

SS

●不能以 disc-PAGE 為唯一分子量證據You can’t take disc-PAGE as your only evidence for MW determination

Mo

l m

ass

7

質譜儀可檢定蛋白質身分

MALDI-TOF

Protease digestion

●比對各片段分子量可確定該蛋白質身分Tryptic fragments identified by MALDI-TOF could identify an unknown protein

m/z

P1 P2 P3 P4

MW4 MW2 MW3 MW1

Unknown protein

Search Database

Candidate protein

Digestion Simulation

MW4 MW2 MW3 MW1

Calculate Mol wt

8

圖 8.1

8.1

.3

■以質譜儀進行蛋白質序列分析

LC

ESI-Mass/Mass

Protease digestion

P1 P2 P3 P4

Unknown protein

P4

GK

GS

WV

R

-GKGSWVR●質譜儀可直接定序Determine the amino acid sequence by ESI/MS/MS (de novo sequencing)

P1 P2 P3

ESI-Mass/Mass

P4

PLC

P4

P

●也可定出磷酸化位置Can also determine the phosphorylation sites

Protease digestion

Phosphorylated protein 9

圖 8.2

產生一系列片段

Ionization(evaporation)

Source

+

Analyzer Detector

Spectrum

Adapted from Liebler (2002) Introduction to Proteomics, p.56, 58

ionsample

MALDI TOF

sample (Matrix Assisted)

++++

+

+ +

+

Time Of Flight

Laser

Ionization

Desorption

■ 質譜儀由三個部分組成 MALDI-TOF 為例10

以雷射激發樣本成為離子形式

樣本附著在 matrix 可保較大片段

離子形式樣本在質譜儀中飛翔

快慢決定於片段的分子量大小

偵測器上可測得

對應質量的片段

m/z

m/z

Source Analyzer Detector

Adapted from Liebler (2002)

Introduction to Proteomics,

p.68 ,70

Quadrupole 四極棒

LC +

+

+

+ ++

+

++ +

+

+high

voltage needle

solvatedions

desolvatedions

ElectroSpray Ionization

-

+

+

(or Ion-trap, Q-TOF)

Triple Quadrupole

ESI MS/MSLC

-

++

++ +

+

+

Q1q2

Q3更小片段挑選片段

鈍氣轟炸

繼續分析

也可以再接 TOF

■ ESI-MS/MS 可分離更小片段11

分析分離樣本

四極棒依照粒子 m/z 篩選

m/z

可有三層四極棒(Q1-q2-Q3)

■ AIV Hemagglutinin 以糖基保護 trypsin sites12

DKICIGYHANNSTAKVDTIMEKNVTVTHAKDIPEKKHNGKLCGLNGVKPLILRDCSVAGWLLGNPMCDEF

LMVPEWSYIVEKNNPVNGLCYPGDFQDYEELKHLLSSTTHFEKIQMFPRNSWPQHDTS_GVTAACPFNGK

_SSFFRNMVWLIKK_NNEYLTIKRGYKNTNQEDLLIMWGIHHPSHDEEQVRLYKNPRTYISVGTSTLNQR

LSPIIAERPQVNGQSGRMSFYWTILKPSDTINFETNGNFIPPEYAFKIVKKGD_SAIIRSELEYGNCNTR

CQTPMGALNSSMPFQNIHPITIGECPKYVKSNRLVLATGLRNIPQIETR

DKICIGYHANNSTTQVDTILEKNVTVTHSVELLENQKEERFCKIMKKGPLDLRECTIEGWILGNPKCDLL

LGDQSWSYIVERPTAQNGICYPGVLNEVEELKALIGSGERVERFEMFPKSTWAGVDTSRGVTNACPSDTI

DSSFYRNLLWIIKTGSAEYPVIKGTYNNTGNQPILYFWGVHHPPDTMVQNTLYGSGDRYVRMGTESMNFA

KSPEIAARPAVNGQRSRIDYYWSVLKPGETLNVESNGNLIAPWYAYKFVSTNKKGAVFKSNLPIENCDAT

CQTIEGVLRTNKTFQNVSPLWIGKCPKYVKSESLRLATGLRNVPQIETR

-

--

-

DKICIGYHANNSTAKVDTIMEKNVTVTHAKDIPEKKHNGKLCGLNGVKPLILRDCSVAGWLLGNPMCDEF

LMVPEWSYIVEKNNPVNGLCYPGDFQDYEELKHLLSSTTHFEKIQMFPRNSWPQHDTS_GVTAACPFNGK

_SSFFRNMVWLIKK_NNEYLTIKRGYKNTNQEDLLIMWGIHHPSHDEEQVRLYKNPRTYISVGTSTLNQR

LSPIIAERPQVNGQSGRMSFYWTILKPSDTINFETNGNFIPPEYAFKIVKKGD_SAIIRSELEYGNCNTR

CQTPMGALNSSMPFQNIHPITIGECPKYVKSNRLVLATGLRNIPQIETR

DKICIGYHANNSTTQVDTILEKNVTVTHSVELLENQKEERFCKIMKKGPLDLRECTIEGWILGNPKCDLL

LGDQSWSYIVERPTAQNGICYPGVLNEVEELKALIGSGERVERFEMFPKSTWAGVDTSRGVTNACPSDTI

DSSFYRNLLWIIKTGSAEYPVIKGTYNNTGNQPILYFWGVHHPPDTMVQNTLYGSGDRYVRMGTESMNFA

KSPEIAARPAVNGQRSRIDYYWSVLKPGETLNVESNGNLIAPWYAYKFVSTNKKGAVFKSNLPIENCDAT

CQTIEGVLRTNKTFQNVSPLWIGKCPKYVKSESLRLATGLRNVPQIETR

-

--

-

-

--

-

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

2838V

3233

329

325

70

70

140

139

210

207

280

276

329

325

70

70

140

139

210

207

280

276

N11 N23

N11 N23

N167

X

N291

N285

N11 N23

N11 N23

N167

X

N291

N285

AIV 2838V病毒外殼蛋白 HA1上面有幾個糖基化位置 (綠色塊)，這些糖基可以保護附近的 trypsin sites (紅色 K, R)，其中 R201在病毒 Receptor binding site (RBS) 附近，是病毒入侵宿主的關鍵。

AIV 3233 在 N167 沒有相對糖基 (X)，因此其感染力減低很多。

RBS

He JL (2015) Virus Research 197:101

R201

■ 以質譜分析檢定蛋白質構造 (AIV HA)13

A 去糖基處理 B 質譜儀分析

PNGase-F - +

2838V

HA1 HA1*

DKICIGYHANNSTTQVDTILE

KNVTVTHSVELLENQKEERFC

KIMKKGPLDLRECTIEGWILG

NPKCDLLLGDQSWSYIVERPT

AQNGICYPGVLNEVEELKALI

ESGERVERFEMFPKSTWAGVD

TSRGVTNACPSDTIDSSFYRN

LLWIIKTGSAEYPVIKGTYNN

TGNQPILYFWGVHHPPDTMVQ

NTLYGSGDRYVRMGTESMNFA

KSPEIAARPAVNGQRSRIDYY

WSVLKPGETLNVESNGNLIAP

WYAYKFVSTNKKGAVFKSNLP

IENCDATCQTIEGVLRTNKTF

QNVSPLWIGKCPKYVKSESLR

LATGLRNVPQIETR

DKICIGYHANNSTTQVDTILE

KNVTVTHSVELLENQKEERFC

KIMKKGPLDLRECTIEGWILG

NPKCDLLLGDQSWSYIVERPT

AQNGICYPGVLNEVEELKALI

ESGERVERFEMFPKSTWAGVD

TSRGVTNACPSDTIDSSFYRN

LLWIIKTGSAEYPVIKGTYNN

TGNQPILYFWGVHHPPDTMVQ

NTLYGSGDRYVRMGTESMNFA

KSPEIAARPAVNGQRSRIDYY

WSVLKPGETLNVESNGNLIAP

WYAYKFVSTNKKGAVFKSNLP

IENCDATCQTIEGVLRTNKTF

QNVSPLWIGKCPKYVKSESLR

LATGLRNVPQIETR

17013095

72

55

43

34

26

2838V

- + PNGase-FkDa

HA1

HA1*

AIV 2838V病毒外殼蛋白 HA1

經 trypsin 水解後以質譜儀分析，

HA1 若先以 PNGase-F 去除其

糖基導致分子量變小 (HA-1*)。

比較 HA1與 HA-1*經質譜儀分析後所得之胜肽片段，

發現 HA-1* 多出三段，其切位落在 K22, R201, K307，

在構造上分別受到 N23, N167, N291 的糖基所保護。

He JL (2015) Virus Research 197:101

(下面粗體字是 MS 有測到的片段)

8.2 蛋白質構造與組成分析 Protein structure analysis

8.2.1 N-端或 C-端胺基酸 Terminal determination

通常都直接定序但 C-端較為困難

8.2.2 胺基酸組成分析 Amino acid composition

8.2.3 胺基酸定序法 Amino acid sequence

8.2.3.1 From cDNA sequence

8.2.3.2 Edman degradation

8.2.4 胜 圖譜 Peptide mapping

8.2.5 其它相關方法 Other methods

Some old techniques become very useful in modern proteomic study

14

決定N端胺基酸

DansylationOnly N-terminal

amino acid modified

HCl hydrolysis

TLC plate

2D

chromatogram

Amino acid hydrolysate

Visualized under UVN-terminal

TLC

TLE

Determine the N-terminal amino acid by dansylation 圖 8.3

15

每種胺基酸的位置都不相同

8.2

.1

或 HPLC

■以薄層層析法鑑定胺基酸

●二次元薄層層析電泳可分離並檢定二十種胺基酸20 amino acids are separated and identified on 2D TLC/TLE

H2O

-F

orm

ic a

cid

2nd dimension

O

1st dim

ensio

n

Dans OH

Arg

Benzene - Acetic acid

TLC

TLE

16

8.2.2 蛋白質酸性水解 Total hydrolysis of protein

● Detection: by HPLC (ion exchange) next slide

● Notice: Some amino acids are destroyed (Trp)

● Condition: 110C, 24 hours, under vacuum

● Reagent: 6 N HCl or 4 N methanesulfonic acid

Cys-Cys broken to Cys

Gln & Asn are acidified to Glu & Asp

(Glu + Gln → Glx; Asp + Asn → Asx)

17

如何測定單獨的 Glu & Gln？

■以液相層析分離鑑定胺基酸

0 10 20 min

Acidic BasicNon-polarIn

tensity

Leu

Asp

Thr

Ser

Glu

Gly

Ala

Cys

Val

MetIle

Tyr

Phe

His Lys

Arg

NH4+

(Pro)

18

決定胺基酸序列

+ PITCPeptide

N-

PTH-

1 2

1PTH-

2

1 2

Amino acidAnalysis(HPLC) Second cycle

Cut off N-terminal amino acid

and recover the remaining peptide

PTH-amino acidcleaved

Edman

Degradation

for amino acidsequencing

+ PITC

圖 8.4

198.2

.3

Tools for proteomic research

Large 2-DE tank

Amino acid sequence analyzer

LC/MS/MS

20

8.2.4 胜 圖譜 Peptide mapping

8.2.4.1 蛋白質專一性水解 Specific proteolysis

專一性內切 Specific endo-peptidase

Trypsin, Chymotrypsin, Sa protease

化學反應法 Chemical method

CNBr (Met)

8.2.4.2 分離胜 的方法 Identify peptides

TLE/TLC HPLC SDS-PAGE

Peptide mapping has become a very important tool in proteomic study

21

■ 蛋白質的專一性水解 Specific proteolysis

使用專一性蛋白Use specific endo-protease

變性蛋白質Protein denatured

Protease Cutting Sites

Specific protease can generate a set of fixed peptides for every protein

22

分別切在不同位置

■ 以傳統胺基酸定序法決定蛋白質序列

N-

+ PITC(Edman degradation)

PTH-

檢定胺基酸種類

Identify each amino acid one by one

依序排出胺基酸序列

Obtain the amino acid sequence of the peptide

由兩組序列推出整段 (比較重疊部份可推出各片段順序)

Compare two sets of fragment sequences and deduce the whole

使用兩種不同的蛋白質水解酵素

Use two specific proteases

每條片段分別定序Sequence every fragment

Peptide to be sequencedProtein to be sequenced

得到兩組片段 Obtain two sets of fragments

The traditional protein sequencing method using Edman degradation

23

圖 8.5

■以雙向層析電泳鑑定胜

血紅蛋白4號片段

Hem

oglo

bin

A

Hem

oglo

bin

S

色析 TLC

電泳 TLE

鎌型血球Sickle cell

24

Linus Pauling

不見了

多出來

Peptide map

Hb S 會聚集成長鏈把血球撐開成鎌刀型

Glu → Val

■ 以傳統胺基酸定序決定蛋白質序列

F. Sanger (Cambridge U)

Insulin 胰島素 (A, B chains)

Nelson & Cox (2000) Principles of Biochemistry (3e) p.142

S S

S

S

S S

+NH3

NH3+

-OOC

Q

G

I

V

E

QC

C

T

SI C S

L

YL

EN

YC

N

COO-

F

S

FV

N

Q

H

LC

G

HLVE

A

L

YLVC

GER

G

FYT

PK

T

B-chain

A-chain

25

(1) 人類第一次解出蛋白質序列 (1958)

(2) 發明 DNA 定序方法 (1980)

兩次 Noble Prizes 都與序列有關

8.3 免疫學工具的利用 Immunological tools

8.3.1 抗原製備 Antigen preparation

8.3.2 免疫流程 Immunization protocol

8.3.3 抗体製備 Antibody preparation

8.3.4 抗体應用 Antibody application

26

8.3.1 抗原的種類 Antigen sources

●巨分子抗原 Macromolecules

Protein, polysaccharide, nucleic acid

●小分子抗原 Small molecules

Conjugated to carrier before immunization

●半抗原 (hapten) aflatoxin, citrinin

Carrier is required

●人工合成胜 Synthetic peptides

Carrier is required

Produce monospecific AbKLHor

BSA

Ag

KLH: keyhole limped haemocyanin (貝類血青素蛋白); BSA: bovine serum albumin

Carrier

27

迴避原物種

(幾十個胺基酸以下)

■ 基礎免疫學 Essential immunology

●免疫系統：先天及後天免疫系統Immune systems (innate and adaptive)

●免疫反應：遭遇→動員→掃蕩→休止Immune response (four stages)

●抗体分子：有兩個專一性抗原結合區Antibody molecule (two specific binding sites)

●單株抗体：只對其專一性抗原決定基作用Monoclonal antibody (very specific reagent)

1 23 4

28

Antigen binding site (Ab)vs.

Antigenic determinant, orEpitope (抗原決定基)

小白鼠免疫流程

Antigen (50 mg/mouse)

Emulsified in 0.5 mL

Freund's Complete Adjuvant

At least three booster shots, same dose in 0.5 mLFreund's Incomplete Adjuvant

Booster shots might be reduced

if TiterMax is use as adjuvant

Titer Determination

Total Bleeding, < 1 mL

Pristane (0.5 mL)

NS-1 Cell (10 6 cells)

Ascites FluidsX mL (X = 1-10)

BALB/c

0

2

4

6

8

10

12

14wk

Trial Bleeding

全採血

腹水

加佐劑製成乳劑+ adjuvant → emulsion

The immunization protocol for antiserum or ascites production 圖 8.6

298.3

.2

免疫球蛋白純化流程

沉澱Precipitation

清洗Washing

透析Dialysis

保存Stock

spin down cells (discard)

+ 2X mL PBS

ammonium sulfate (AS)fractionation 0~40% sat.

spin down pellet

spin down pellet

resuspended in 40% AS

spin down precipitate

dissovled in X mL PBS

dialysis in PBSthree changes

+ glycerol (equal volume)

Pellet

Pellet

Supernatant

Ascites or serum (X mL)

IgG (stored in freezer)圖 8.6

308.3

.3

8.3.4 抗体應用 Applications of Ab

a. 轉印及免疫染色法 Western blot & immunostaining

應用最廣且最有效率 (包含細胞組織切片免疫染色)

b. 免疫沉澱法 Immunoprecipitation (pull-down)

另一種檢定專一性抗原的方法

c. 親和層析法 Affinity chromatography

最快速有效的純化方法

d. 雙向免疫擴散法 Double diffusion

古老但仍有其特色及應用

e. 酵素免疫分析法 Enzyme immunoassay

可分析大量樣本 (ELISA)

f. 抗体晶片 Antibody chip

專一快速地同時進行多種分析

31

■雙向免疫擴散法

Outer wells: Rice (R) and maize (M) sucrose synthase (Ag)

Central well: Antiserum against rice sucrose synthase (Ab)

由沉澱線交叉情形可推測抗原分子間的包含關係The crossing-over of the precipitin lines reveals the structural

relationship between the antigen molecules

spur

Ab

R

M

32

+ Substrate

ELISA Plate (solid phase)

Antigen

SpecificAb

2nd Ab

Conjugated enzymeColor change

Non-specific Ab

2

E

EL

ISA

To detect the Ab in the sample by ELISA

33

ELISA幾乎無所不在！

ELISA 操作注意：

(1) 把握 pipetting 及 timing 精準

(2) 確定 2nd Ab conjugate的品質

(3) 檢查樣本中可能的干擾物質

(4) 注意反應液在保溫箱會蒸發

(5) 使用規格正確的 ELISA plate

(6) 設計好關鍵的 control 對照組

■酵素免疫分析法

A

A

■擔体免疫沈澱的原理及應用

A

A

誘生抗体 Ab induction

細胞粗抽取液Cell lysate

AbProtein A-Sepharose

Conjugation

Ag-Ab reaction

Spin down Washing

免疫吸著劑Immunosorbent

Solid support-based immunoprecipitaion 圖 8.7

34

A

A

■擔体免疫沈澱的原理及應用

擔体免疫沈澱Immunoprecipitation

SDS-PAGE

抗原可能有兩個次体 ★Ag might contains two subunits

抗体有輕鏈及重鏈Ab contains H & L chains

H

L

細胞粗抽取液Cell lysate

35

圖 8.7

若抗原的分子量與H或 L重疊怎麼辦？

SDS-PAGE

2DE

Protein ID Match peptide

Adenosylhomocysteinase IVLTIIR

DSAAVFAWK

HSLPDGLMR

LVGVSEETTTGVK

Histone H4 (wheat) IFLENVIR

IDGLIYEETR

TVRAMDVVYALKR

Fructose bisphophate aldolase VTPEVIAEYTVR

IGPNEPSQLAIDLNAQGLAR

Triosephosphate isomerase TNVSPEVAESTR

VIACVGETLEQR

NAD-dependent malate

dehydrogenase

DDLFNINAGIVK

Histone H3 ASAPATGGVK

Putative lipase DQVLEEVRR

AgAE6C3M 1 2

LC/MS/MSAg

Interacted proteins

■ 抗体免疫沈澱與蛋白質交互作用

Interacted proteins

Pull down proteins interacted with Ag

Ag

36

最大的問題：必須驗證何者是有意義的交互蛋白質或者只是非專一性的吸附

要證明細胞中也有交互作用

(How?)

L-SP

Anti-L-SPAnti-

Proteasome

L-SP

H

L

Proteasome

ImmunoprecipitationImmunoprecipitation

L-SPL-SP

ImmunostainingImmunostaining

Anti-Proteasome

Anti-L-SP SDS-PAGE

Western blot

Proteasome

■ 免疫沈澱證明分子間結合 Protein interactions

Western blot

Immunoprecipitation is useful in detecting the interaction between two proteins

37

■ 雙向免疫擴散 Double diffusion works

L-SP

L-SP

L-SP

L-SP+

L-SP

L-SPL-SP

L-SP+L-SP+

L-SP

Control

HX

38

組織切片

39

Merge (red & green)ProteasomeL-SP

DAPI (nucleus)Light Merge (all)

L-SP

Confo

cal m

icro

scop

y

■共軛焦顯微定位

L-SP

Antibody Concentration (pg/spot)

0 20 40 60 80 100 120 140 160 180 200 220

Inte

gra

ted I

nte

nsity (

IOD

)

0

2000

4000

6000

8000

10000

12000

奈米中心林啟萬教授實驗室蛋白質晶片試製

Schleicher & Schuell

E

Nitrocellulose

Juang RH (2005) EPA

Making

protein chips

40

8.4 蛋白質新世界 Protein New World

8.4.1 微量科技 Microscale technology

8.4.1.1 微量純化

8.4.1.2 微量分析

8.4.2 蛋白質体學 Proteomics

8.4.2.1 如何看待 Proteomics？

8.4.2.2 Proteomics 就是蛋白質化學？

8.4.2.3 Proteomics 與代謝体

蛋白質科技Protein Technology

41

(1) 基本的蛋白質生化學沒有改變

(2) 自動化與大數據強化分析平台

(3) 樣本處理極微量且數目巨量化

Protein Chemistry100 years

Purification Analysis

■蛋白質科技相關課程與階段 Related courses

ProductionEngineeringFunctionStructureAnalysisPurification

Basic Protein Techniques

Structure Function

Protein Structure & Function

Protein Eng Biochem Eng

Protein Engineering

蛋白質化學Protein chemistry

生物化學實驗

Biochem lab

酵素化學Enzyme

biochemistry

酵素化學實驗

Enzyme biochemlab

生物質譜學Mass spectroscopy

生物質譜實驗Mass

spectroscopy lab

應用微生物學

Applied microbiology

生化工程學Biochemicalengineering

生化工程實驗

Biochemicalengineering lab

蛋白質工程Protein engineering

蛋白質工程實驗

Protein engineering lab

蛋白質体學Proteomics

蛋白質体學實驗

Proteomics lab

抗体工具Ab tools

生物資訊實驗

Bioinformatics lab

生物資訊學Bioinformatics

結構生物學Structural biology

蛋白質構造與功能

Protein structureand function

生物物理化學Biological

physical chem

42

代謝体學

Metabolomics

Image

analysis

MALDI-TOF

LC/MS/MS

■蛋白質的微量分離及檢定

1 電泳及轉印Electrophoresis and transfer

2 二次元電泳2D electrophoresis

3 膠体內分析In-gel digestion & analysis

4 微量分離純化Micropurification and analysis

微量分析系統Microanalysis

抗体製備Ab preparation

生物資訊學Bioinformatics

蛋白質純化分析新貌A new look for EPA

蛋白質科技Protein Technology

Protein

extraction

Protein

sequencing

Amino acid

sequence

Bioinformatics

database

Protein

transfer

Protein

band

Mass

spectroscopy

LC orelectrophoresis

2D

electrophoresis

In-gel

digestion

Homogeneous

protein

Fast

LC

Capillaryelectrophoresis

Synthetic

peptides

Ab

preparation

43

(假如實驗課的經費無上限)

精準高效純化

微量快速分析

細菌的基因很小，容易被解碼，也有其演化優勢

『細菌的染色体非常進，

其生理功能也很直接...

是非常務實的基因』

由基因密碼可解讀代謝途徑

Celera

並找出攻擊點

基礎代謝圖

J. Craig Venter Institute (2010)

電腦設計出來的微生物Mycoplasma laboratorium [黴漿菌]

有浮水印：VENTERINSTITVTE

以黴漿菌的外殼置入人造染色体

葡萄糖 主代謝路徑

X

■由基因解碼到代謝体學 Decoding the genome

WIKIPEDIA

44

也無法預測蛋白質間的交互作用Nor can you predict the protein interactions

基因表現不一定完全反映到蛋白質Gene expression is not totally reflected in protein level

由基因体較難預測蛋白質的修飾及調控It is difficult to predict the protein modification and regulation from genomic level

Proteome is much complex than its genome

45

pH 3 - - - - - - 10(1) IEF

等電焦集電泳

(2)

SDS-PAGE分離膠体

(3)

Staining染色脫色

■ 二次元電泳操作 2-DE operation

圖 7.9

46

↑ 注意 pH 梯度的產生 ↑

↑ SDS 及加熱處理 ↑

■蛋白質体可綜觀蛋白質的消長與身分

Proteolytic

digestion

GCG

Proteolytic fragmentsPure protein2D electrophoresis(Identify significant difference)

Sample

MALDI-TOF

CapillaryElectrophoresis

HPLC

N-

Amino acid sequencing

Mass spectrum

Databasesearching LC/MS/MS

2D tool provides insight from comparing proteomic difference

47

圖 8.8

■ 微流体平台 Microfluidics, Lab-on-a-chip

所有蛋白質純化與活性分析均微小化

Agilent 2100 bioanalyzer

Agilent HPLC-Chip/MS

前處理pretreatment

分離管柱

質譜儀分析Mass analysis

樣本 sample well

毛細管電泳Capillary electrophoresis

偵測

detection

http://www.chem.agilent.com/Scripts/Phome.asp

Minimize protein purification and analysis in one chip

48

■ 現代蛋白質科技特點 Modern protein technology

●產能 High-through put

●快速 High-speed

●微量 Micro-scaled

49

Antibody是最方便取得之專一性探針

50

Think big!

-omics

驚奇的酵素活性之根本

52