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Protein sample 1 label with Cy3
Pooled internal standard label with Cy2
Protein sample 2 label with Cy5
Mix labelled samples
2-DE separation
Cy5
Fluorescence Scanner with Variable Mode
Cy3 Cy2
DeCyder Differential
Analysis Software
2-D Difference Gel Electrophoresis
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DeCyder Differential Analysis Software
Sample 3 Sample 4
Internal Std GEL B
Sample 5 Sample 6
Internal Std GEL C
Sample 1 Sample 2
Internal Std GEL A
DIA In-gel co-detection
Cross-gel matching
Protein difference ratios
DIA DIA
Master
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Post-Translational Modifications directly detected by special staining
• Important for biological processes, particularly signal transductions
• Most cellular processes are regulated by reversible phosphorylation of proteins
• Studies of phosphorylated proteins involve radioisotope-labeling and specific
antibodies
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Glycoprotein Gel Stain Glycoprotein Gel Stain
CandyCane glycoprotein molecular weight standards containing alternating glycosylated and nonglycosylated proteins were electrophoresed through a 13% polyacrylamide gel. After separation, the gel was stained with SYPRO Ruby protein gel stain to detect all eight marker proteins (left).
Subsequently, the gel was stained by the standard periodic acid–Schiff base (PAS) method in the Pro-Q Fuchsia Glycoprotein Gel Stain Kit to detect the glycoproteins alpha2 -macroglobulin, glucose oxidase, alpha1-glycoprotein and avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate) Molecular Formula: C15H18Cl2N3O5P (MW 422.20) Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
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Only SYPRO Ruby
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Pro-Q Diamond Staining of a 2-D gel
• Selectively stains phosphoproteinsin 2-D gel
• Allows direct in-gel detection of phosphate groups attached to tyrosine, serine, or threonine residues
• NO NEED for antibody and radioisotope
• Signal correlates with the number of phosphate groups
• Fully compatible with mass spectrometry
• Ratio of Pro-Q diamond to Sypro Ruby
=phosphorylation level/total protein
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Phosphoprotein
Phosphoprotein Stain Stain
Visualization of total protein and phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell lymphoma line cell lysate were separated by 2-D gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (blueblue) followed by SYPRO Ruby protein gel stain (redred). After each dye staining, the gel was imaged and the resulting composite image was digitally pseudocolored and overlaid.
T.H. Steinberg et al., Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144
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http://www.kendricklabs.com/pric.htm#Staining From sample preparation to 2-D analysis service
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Robot Spot Cutter
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Trypsin digest Spot cutting
MALDI spotting
MALDI-Q-TOF
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影像比對及相對定量分析
影像比對及相對定量分析
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挖取變異點 挖取變異點
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挖取變異點 挖取變異點
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蛋白質膠內水解 蛋白質膠內水解
回溶
回溶乾燥
乾燥Peptide Peptide 與膠體分離
與膠體分離蛋白質水解酵素
蛋白質水解酵素消化
消化退染
退染去除雙硫鍵
去除雙硫鍵272
In-Gel digestion Day 1
穿上實驗衣及手套,且袖口需用膠帶固定(防紫外線照到),把gel放在UV box上,
1. Transfer the gel slice into a 650μl tube (siliconized這是要特別指名, methanol washed). It is better to cut gel into 1mm2size.
2. Reduction / Alkylation(本步驟藥品需現配 不可使用stock)
z Add 100ul of 65mM Dithioerythritol (DTE) /25mM ammonium bicarbonate (pH 8.5) into sample gel. Soak for 1 hour at 37℃.
z Centrifuge 10000g 1min . Remove DTE completely.
z Add 100ul of 100mM Iodoacetamide (IAA) / 25mM ammonium bicarbonate (pH 8.5) into sample gel. Soak for 1 hour at R/T at dark.
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zCentrifuge 10000g 1min . Remove IAA completely.
1.Add 100 ul of 50% acetonitrile / 25mM ammonium bicarbonate buffer (pH to 8.5). Allow gel to soak for 15mins. Centrifuge at 1000g for 1 minute. Remove buffer completely. Repeat this step twice (Depends on intensity of the dye,直到膠沒有顏色為止). Destain
2.Soak the gel in 100% acetonitrile (膠會變成白色的), and centrifuge at 1000 g for 1 min. Remove acetonitrile.
3.Dry the gel slice for 5mins in a speed vac. at R.T.
4.Rehydrate the gels with: Coomassie stain- 10ul of trypsin in 25mM ammonium bicarbonate (pH 8.5) to a final of 0.1ug. Sypro Rube stain-10ul of trypsin in 25mM ammonium bicarbonate (pH 8.5) to a final of 0.0225ug. (Trypsin amount provide here was recommended. Actual trypsin amounts are highly protein dependent.) 如果trypsin 太高會影響 MS結果
Crash the gel slice with siliconized blue stick. If trypsin solution does not cover gel pieces, add more 25mM ammonium bicarbonate (pH8.5) to cover all gel pieces. 用特殊siliconize的棒子,往底部轉三下,把膠弄散但不要太 碎(水),這樣
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• END
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Central tool for proteomics
Developed in 1975 (O’Farrell and Klose)
Requirement: sample from different subjects (control and test; treated and non test; ); Several gels and high repetitive
Computer analysis
Statistical tool: variation up to 40% between a gels of a same sample → produce 3-5 gels from identical material for better results
2-DE in proteomics
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Main approaches to 2-DE analysis
Creation of proteome maps: systematic analysis of all spots on a gel---- one gel, and all proteins
Monitoring of gene product over a set of 2-DE gels--- set of gels, selected proteins
Differential expression of proteins: global investigation between 2-DE image of several populations---several gels, differential analysis
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Some common trouble in 2DE
IPG strip rehydration not enough IPG strip length rehydration buffer
7 cm 125 ul 11 cm 185 ul 17 cm 300 ul 18 cm 315 ul 24 cm 410 ul
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Some common trouble in 2DE
Bubbles between IPG and SDS-PAGE
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Some common trouble in 2DE
Protein precipitation
1.Rehydration buffer 2.Protein ↓
3. IEF start voltage-time (may be 塞車)
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Protein too high
Some common trouble in 2DE
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Some common trouble in 2DE
1.SDS-PAGE pH wrong 2.Strip equilibrium time less 3.SDS concentration to less
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Some common trouble in 2DE
IEF X
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Some common trouble in 2DE
DTT 量不足,造成鹼 性區域出現横條
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Some common trouble in 2DE
Many interference substanceLipid, DNA, RNA, salt, …
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Sample preparation x
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No high molecule weight protein
Protein degradation
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Stain and destain
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SDS-PAGE not good
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Examples of 2-DE results
D
Healthy control Patient
Digest to peptide fragment MS analysis
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Important molecular biology techniques/assays
(Ch. 5, pg. 91-131)1) Gel electrophoresis, Pulsed field gel electrophoresis 2-dimensional gel electrophoresis
2) Denaturing gel, Native gel, SDS-PAGE, coommassie staining 3) Chromatography - ion exchange and gel filteration
4) Autoradiography, Phosphorimaging, Scintillation counting 5) Nucleic acid hybridization - Southern, Northern , Western blot 6) DNA fingerprinting and DNA typing
7) DNA sequencing - manual and automated 8) Restriction mapping
9) Site directed mutagenesis
10) S1 mapping, Primer extension, Run-off/run on transcription, &
G-less cassette, reporter gene (CAT, luciferase, GFP) 11) Filter binding, Gel mobility shift assays
12) DNase I footprinting assay 13) Co-immunoprecipitation assay 14) Yeast two-hybrid assay 15) Knockouts
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Run 2-DE step by step
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SDS procedure
In 2-DE analysis, for emergency case.
Have to dilute SDS samples at least 20 fold with urea an a non or zwitterionic detergent containing solutions.
The major reasons for using SDS:
1.
Formation of oligomers can be prevented2.
Dissolved tough cell walls samples (withboiling)
3.
Dissolved very hydrophobic proteins Usually, in western blot anlaysis300