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蛋白質體學
Proteomics 2011
Protein Sample Preparation
for Proteomic Research
陳威戎
2011. 09. 26
References
1. Enzyme Purification and Analysis (2005),
Juang R. H. (台大生化科技系 莊榮輝老師)
2. Principles of Proteomics (2004),
Twyman R. M. ed.
3. Ettan Sample Preparation Manual –
Right from the start, Amersham
Biosciences
4. 蛋白質體學 (2003)
教育部顧問室生物技術科技教育改進計畫 補助
醫藥基因生物技術教學資源中心 主編
Proteome Analysis and Proteomics
Proteomic Approaches
With Proteomics to Quicker Results
Genomics and proteomics: high throughput
Workflow of proteomic research
Major equipment for proteomic research
蛋白質體研究的挑戰 Challenges of proteomics
Proteomics Technology
蛋白質樣品製備 Sample preparation in proteomics
Right from the start: Preparation is everything
The first step to generating reliable, accurate
results is getting the first step right.
Sample preparation is crucial to obtaining good
data.
Key proteins lost during initial sample
preparation can never be restored.
Sample preparation- right from the start
 蛋白質樣品抽取 Sample extraction
 蛋白質樣品溶解 Sample solubilization
 蛋白質樣品分劃 Sample fractionation
 蛋白質樣品清潔 Sample clean-up
 細胞裂解與均質 Cell lysis and homogenization
 其他污染物移除 Removal of other contaminants
 防止蛋白酶水解 Prevention against proteolysis
蛋白質樣品抽取 Sample extraction
 如何開始？ How to start?
5W principles 基本原則
 材料來源 Materials & sources
材料取得與保存
 均質及抽取 Homogenization & extraction
確實做好第一步
Juang RH (2005) EPA
■ 如何開始？ How to start?
先考慮以下諸點：
5W
a. 要純化那一個蛋白質？ What ?
b. 為何要純化此蛋白質？
c. 由何種材料純化？
d. 由那一個生長期？
e. 如何純化此蛋白質？
Why ?
Where, from ?
When ?
How ?
Juang RH (2005) EPA
■ 酵素蛋白質純化過程的三個階段 Three stages
(1) 粗蛋白 (crude protein)︰
採樣 → 均質打破細胞 → 抽出全部蛋白，
多用鹽析沉澱法。
(2) 部分純化 (partially purified)︰
初步的純化，使用各種管柱層析法。
(3) 均質酵素 (homogeneous)︰
目標酵素的進一步精製純化，可用製備式電
泳或 HPLC 等。
Juang RH (2005) EPA
A
(1) Crude Protein (1% pure) (2) Partially Purified
(50~90% pure)
(3) Homogeneous (>99%)
■
酵
素
純
化
階
段
及
分
析
方
法
B
Purification
Material
Analysis
Background knowledge about Material
Protein / Activity assay
(1) 粗蛋白
Extraction
Crude protein
Electrophoresis
Protein
fractionation
Naive-PAGE
SDS-PAGE
Gradient PAGE
Ammonium sulfate
Organic solvent
Kinetic study
Molecular weight
Sedimentation coefficient
determination
Quaternary structure
Isoelectric focusing
Peptide mapping
Chromatography
Gel filtration
Ion exchange
Affinity chromatography
FPLC
Electrophoresis
(2) 部分純化
Partially purified
Preparative electrophoresis
Isoelectric focusing
Amino acid analysis
Protein sequencing
Extinction coefficient
Pure Enzyme
Antibody production
Monoclonal or conventional
Crystal
X-Ray
crystallography
Spectrometric methods
Immunoassays
Immunoblotting
ELISA
Double diffusion
Immunoelectrophoresis
(3) 均質酵素
Homogeneous
CD, ORD, NMR & ESR
Juang RH (2005) EPA
■ 各種純化或分析方法的原理
Cell
Cell homogenization
Small molecules
Organelle
separation?
Macro molecules
Amino acid,
monosaccharide, Nucleic acid
nucleotide, fatty acid
Protein
Ammonium sulfate
Polysaccharides
細胞
碎片
Cell debris
precipitation
Molecular size
Molecular charge
Molecular polarity
Gel filtration,
SDS-PAGE,
Ultrafiltration
Reverse phase
Ion exchange,
Affinity
chromatography,
Chromatofocusing,
chromatography,
HIC,
Disc-PAGE,
Hydroxyapatite
Salting-out
Isoelectric focusing
Basic protein properties useful for planning separation steps
Affinity
Juang RH (2005) EPA
蛋白質樣品抽取 Sample extraction
 如何開始？ How to start?
5W principles 基本原則
 材料來源 Materials & sources
材料取得與保存
 均質及抽取 Homogenization & extraction
確實做好第一步
Juang RH (2005) EPA
■ 目標材料之選擇 What’s your starting material?
 Which organism?
動物、植物、微生物
 Which tissue?
根莖葉花果或組織培養
 Which organelle?
細胞核、液泡、葉綠体
 Secreted enzyme?
有關酵素的穩定性
 Membrane protein?
影響抽取策略的設計
Juang RH (2005) EPA
St Pierre,B.; Bertrand,C.; Camirand,A.; Cappadocia,M.; Brisson,N. (1996) Plant Molecular Biology 30: 1087-1098
The starch phosphorylase gene is subjected to different modes of regulation in starch-containing tissues of potato
SP RNA expression (potato tissues)
SP protein expressed (potato tissues)
A typical plant cell
Buchanan et al (2000) Biochemistry and Molecular Biology of Plants p.3
■
一
個
典
型
植
物
細
胞
■ 植物的細胞壁 Plant cell wall is a barrier
● Old plant cell wall is very tough to break
Buchanan et al (2000) Biochemistry and Molecular Biology of Plants p.54
■ 植物色素在純化上的問題 Pigment is a problem
● Pigment stains the gel during purification
Scientific American
■ 細胞膜蛋白質抽取較困難 Membrane proteins
● Detergent (Triton X) is used to solubilize membrane proteins
Polar
Non-polar
Buchanan et al (2000) Biochemistry and Molecular Biology of Plants p.8
■ 植物材料問題 Special problems for plant materials
 細胞壁 Cell wall 較難打破
 葉綠体 Chloroplast 特有的酵素
 液 泡 Vacuole 有許多干擾物質
蛋白酶 (proteases)
多酚化合物 (polyphenols):
Alkaloid
生物鹼
Flavonoid 類黃酮
Tannin
單寧
Juang RH (2005) EPA
■ 植物色素之產生與去除 How to remove pigment
b-mercaptoethanol
inhibit
Phenol oxidase
Decrease enzyme catalysis
Low temperature
Oxidation
Phenolic compound →→→ Pigment
adsorb
吸附
Polyvinylpolypyrrolidone
(PVPP)
Juang RH (2005) EPA
蛋白質樣品抽取 Sample extraction
 如何開始？ How to start?
5W principles 基本原則
 材料來源 Materials & sources
材料取得與保存
 均質及抽取 Homogenization & extraction
確實做好第一步
Juang RH (2005) EPA
Sample Preparation
Cell disruption methods
Cell lysis and homogenization
The first step in protein analysis is the disruption of
cells or tissues, and homogenization.
The method used to isolate proteins from intact cells
and tissues should
- minimize loss of proteins, especially membranebound proteins
- maximize recovery
- retain the structural integrity of the protein
- avoid introducing new contaminants
- concentrate the sample to optimal detection range
打破細胞的方法 101 ways to break the cell
Dry way:
液態氮研磨 (grinding in liquid nitrogen), 磨粉
機 (coffee grinder), 球磨機 (ball mill)
Wet way:
均質器 (homogenizer), 果汁機 (Waring blender),
Polytron, 研砵 (mortar), 玻璃球 (glass bead mill),
超音波震盪 (ultrasonication), French press
Is your target protein released from the cell?
Juang RH (2005) EPA
Mathews et al (1999) Biochemistry (3e) p.299
 某些胞器的分離相當困難 Some organelles are difficult to isolate
▼ Homogenizer for breaking the
cell but leave organelles intact
Alberts et al (2002) Molecular Biology of the Cell (4e) p.794
研磨樣本常用方法 Two popular methods
● Use Polytron
Various size of probe
● Use liquid nitrogen
Juang RH (2005) EPA
細胞打破之後 After breaking the cell…..
(1) 降低溫度
Keep temperature low
(2) 儘速純化
Purify as soon as possible
(3) 避免氧化
Avoid oxidation
(4) 避免吸著
Avoid adsorption by flask
(5) 避免污染
Avoid contamination
Juang RH (2005) EPA
蛋白質樣品溶解 Sample solubilization

Neutral chaotropes

Urea (8~9 M)
Urea (7 M) + thiourea (2 M)

2-mercaptoethanol
Dithiothreitol (DTT)
Dithioerythritol (DTE)
Tributylphosphine (TBP)
Neutral or zwitterionic
detergent
Triton X-100
Nonidet P-40
CHAPS (2~4%)
SBS-10
(SDS ?)
Reducing agents for
disulfide bonds

Ampholytes and buffer
Ampholyte: 2% (v/v)
Buffer: 10~100 mM
Reducing agents
Protein Precipitation
Protease Inhibitors
Desalting techniques
Nucleic acids removal
Ettan Sample Preparation Kits
蛋白質樣品清潔 Sample clean-up
 Protein precipitation is often applied prior to 2-DE
to:
1. selectively separate proteins from
contaminating substances.
2. concentrate proteins from samples that are too
dilute for effective analysis.
 Incomplete protein precipitation results in
significant loss of total protein from the sample,
introducing a bias to the result.
Sample clean-up~ How it works?
 Uses precipitant and coprecipitant in
combination to quantitatively precipitate the
sample proteins.
 These proteins are pelleted by centrifugation and
the precipitate is further washed to remove nonprotein contaminants.
 After a second centrifugation, the resultant pellet
is resuspended into denaturing sample solution
for first-dimension IEF.
Sample clean-up eliminates horizontal
streaking caused by residual SDS
Outer membrane protein F-precursor was
successfully identified after sample clean-up
Efficient dialysis with Mini-Dialysis
Easy-to-use compared to dialysis bags
Designed for efficient dialysis of small
sample volumes
Conical tube bottom maximizes sample
recovery
Various choices for efficient dialysis based
on MW cut-off and volume
Improves resolution in 2-D gels
Sample dialysis using Mini-Dialysis
Effect of dialysis on 2-D resolution sample
Albumin and IgG removal
Proteins in serum and other biological fluids are
difficult to resolve by 2-DE, largely due to the
abundance of serum albumin and IgG.
Serum albumin: 50-70% ; IgG: 10-25%
Obscure other proteins in the gel, and limit the
amounts of proteins in the serum that can be resolved
by 2-DE.
Have wide pI and MW ranges that further reduce
resolution and mask other low-abundance proteins.
Schematic of the removal process
Removal of albumin and IgG from human serum
Removal of albumin from human serum
Removal of other contaminants
 Organic/Inorganic
molecules
- TCA/Acetone
precipitation
- Organic solvent
precipitation
- Wash away PBS
 Nucleic acids
- Nuclease treatment
 Lipids
- Organic solvent
precipitation
- Excess detergents
 Phenolic compounds
- Reducing agents
(DTT, DTE)
- Organic solvent
precipitation
Prevention against proteolysis
 Chaotropes of high concentration
- 8 M Urea
 Protease Inhibitor
- PMSF and AEBSF
- EDTA or EGTA (< 1 mM)
- protease peptide inhibitor
- protease inhibitor mix (cocktail)
Typical sample preparation for 2DE
 A urea-based solution + nonionic detergents +
reducing agents + a protease inhibitor
(O’Farrell’s lysis buffer: 9M urea, 4% NP-40, 50-100 mM DTT)
(modified lysis buffer: 9M urea, 2-4% CHAPS, 1% DTT,
2% carrier ampholytes)
 Solubility improvement: thiourea, tributylphosphine,
zwittergents
 TCA/Acetone precipitation  solubilization by lysis
buffer
 SDS boiling  diluted by lysis buffer