8 蛋白質科技 protein technology - 國立臺灣大學juang.bst.ntu.edu.tw/epa/pdf/2018s ana...
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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