99 年台灣大學暑期生物技術研究班 蛋白質體學技術簡介 輔仁大學生命科學系 陳翰民 Common techniques in proteomics Separation of proteins 1DE (convention) 2DE (modern) Multi-dimensional HPLC (modern) Analysis of proteins Edman Sequencing (convention) Mass Spectrometry (modern) Database utilization
About the old techniques The pioneer of electrophoresis The Nobel Prize in Chemistry 1948 Arne Wilhelm Kaurin Tiselius was born August 10,1902, in Stockholm. After the early loss of his father,the family moved to Gothenburg where he went to school, and after graduation at the local "Realgymnasium" in 1921, he studied at the University of Uppsala, specializing in chemistry. He became research assistant in The Svedberg's laboratory in 1925 and obtained his doctor's degree in 1930 on a thesis "The moving-boundary method of studying the electrophoresis of proteins" (published in Nova Acta Regiae Societatis Scientiarum Upsaliensis, Ser. IV, Vol. 7, No. 4) and was appointed Docent (Assistant Professor) in Chemistry from 1930 on.
Operation of SDS-PAGE cathode Anode SDS-PAGE Poly-acrylamide gel electrophoresis Has not changed in the 32 years since its inception..the question answered is still the same Resolution depends on gel length Most convenient technique to separate protein
Edman degradation Introduced in 1949 1. Run PAGE 2. Digest whole protein mixture and start sequencing Only works with high abundance proteins Edman degradation 1953, Frederick Sanger worked out the sequence of insulin 1958 Nobel Prize in Chemistry 1980 Nobel Prize in Chemistry: DNA sequencing
Edman degradation Only identify the first a.a. CN061002-Chen4 Standard 1 QT 57.00 54.00 D N S G E H A R Y P d-ptu W K 51.00 48.00 3.0 6.0 9.0 12.0 15.0 18.0
CN061002-Chen4 Residue 1 57.00 d-ptu F 54.00 51.00 K 48.00 D N S E H A R Y W 3.0 6.0 9.0 12.0 15.0 18.0 CN061002-Chen4 Residue 2 57.00 54.00 M d-ptu 51.00 A K 48.00 D N E H R 3.0 6.0 9.0 12.0 15.0 18.0
CN061002-Chen4 Residue 3 57.00 K 54.00 d-ptu 51.00 A M V 48.00 D N 3.0 6.0 9.0 12.0 15.0 18.0 CN061002-Chen4 Residue 4 57.00 54.00 N d-ptu 51.00 48.00 D G E A K 3.0 6.0 9.0 12.0 15.0 18.0
CN061002-Chen4 Residue 5 d-ptu 57.00 54.00 G 51.00 D N A F K 48.00 3.0 6.0 9.0 12.0 15.0 18.0 CN061002-Chen4 Residue 6 57.00 54.00 A d-ptu 51.00 G 48.00 D N 3.0 6.0 9.0 12.0 15.0 18.0
CN061002-Chen4 Residue 7 57.00 54.00 d-ptu K 51.00 A D N G W 48.00 3.0 6.0 9.0 12.0 15.0 18.0 CN061002-Chen4 Residue 8 57.00 54.00 d-ptu 51.00 T D N Y 48.00 3.0 6.0 9.0 12.0 15.0 18.0
CN061002-Chen4 Residue 9 d-ptu 57.00 54.00 I 51.00 T K D N M V 48.00 3.0 6.0 9.0 12.0 15.0 18.0 CN061002-Chen4 Residue 10 57.00 54.00 d-ptu 51.00 G I K 48.00 D N 3.0 6.0 9.0 12.0 15.0 18.0
Technology, Now and then Traditional RNA technique : Northern blotting Isolated RNA Electrophoresis Blotting Probing Labelling on probes!! Developing 1. Estimated time to get results: 2-3days 2. Expressed Gene (mrna) checked: 1-8 species 3. Accuracy: Low to moderate
High-throughput method: Microarray Labeling on sample mrna as probe cdna or oligonucleotide spotted on chips Clustered genes data analysis Clustered experiments 1. Estimated time to get results: 5-7 days 2. Expressed Gene (mrna) checked: thousands 3. Accuracy: moderate to high Traditional Protein technique: protein purification and edman degradation Cut desired band Database searching for homolog Peptide N terminal sequencing 1. Protein purification: necessary 2. Protein idetified: 1 per purified sample 取材自台大微生物生化系莊榮輝教授網頁
High throughput technique: 2D electrophoresis + Mass spectrometry 1. Protein purification: not necessary 2. Protein idetified: up to thousands per unpurified sample 取材自台大微生物生化系莊榮輝教授網頁 Major techniques in modern proteomics A. Two dimensional electrophoresis, 2-DE B. Mass spectrometry
Separation identification 取材自台大微生物生化系莊榮輝教授網頁 The pioneer of two dimensional electrophoresis Patrick H. O'Farrell, PhD Professor, Biochemistry and Biophysics, UCSF O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007. A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources.
The pioneer of two dimensional electrophoresis The Nobel Prize in Chemistry 2002 John B. Fenn is the chemist who invented the electrospray method. Today it is used in laboratories all over the world. Fenn has worked mainly as Professor of Chemistry at Yale University, USA, and at Virginia Commonwealth University, USA. Koichi Tanaka worked as a research engineer at Shimadzu Corp. in Kyoto, Japan. Tanaka's idea was to use the energy from laser light, ingeniously transferred to the proteins, to get them to let go of one another and hover freely. It worked! Automation process for 2-DE + mass spectrometry Image Analysis Gel hotel Spot picker 2-D Gel Digester MS Spotter
The old way to find protein markers Takes months to years Sample homogenization Prepare for chromatography Run Chromatography Prepare for Chromatography again Run Chromatography again XT C1 C2 C3 cut? Run SDS-PAGE and Transfer to membrane Sequence N termini (Edman degradation) Search database for homologous match What s proteomics? "The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type. Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium. Electrophoresis 16 (1995) 1090-1094 Two MOST applied technologies: 1. 2-D electrophoresis: separation of complex protein mixtures 2. Mass spectrometry: Identification and structure analysis
The new way to find protein markers 取材自台大微生物生化系莊榮輝教授網頁 How proteomic techniques help? Identification of protein markers from patient samples patient Mass Healthy people Global profiling proteomics
The identities of proteins The ID for proteins Name: hemoglobin MW: 20kDa pi: 5.67 Hydrophobicity: -20? Size: molecular weight (utilized in 2-DE) Charge: pi (utilized in 2-DE) Hydrophobicity Two dimensional electrophoresis (2-DE) 1. First dimension: denaturing isoelectric focusing separation according to the pi 2. Second dimension: SDS electrophoresis (SDS-PAGE) Separation according to the MW Interested spot Digest to peptide fragment MS analysis
Run 2-DE, step 1 Run 2-DE, step 2
Run 2-DE, step 3 Run 2-DE, step 4
The theory of IEF The IEF is a very high resolution separation method, and the pi of a protein can be measured. How ph gradient forms, Immobilized ph gradient, IPG First developed by Righetti,(1990). Immobilized ph gradient generated by buffering acrylamide derivatives (Immobilines) Immobilines are weak acid or weak base. General structure CH 2 CN C N R CH 2 CN C N H O H O H R = amino or carboxylic groups Acrylamide
Schematic drawing of IPG matrix First dimension electrophoresis instrument Amersham Biosciences Bio-Rad
Run IEF, STEP 1 1. Remove protective film from Immobiline DryStrip gel. Run IEF, STEP 2 2. Apply rehydration solution to the Strip Holder.
Run IEF, STEP 3 3. Wet entire length of IPG strip in rehydration solution by placing IPG strip in strip holder (gel facing down). Run IEF, STEP 4 4. Gently lay entire IPG strip in the strip holder, placing the end of IPG strip over cathodic electrode.
Run IEF, STEP 5 5. Protein sample can be applied at sample application well following the rehydration step if the protein sample was not included in the rehydration solution. Run IEF, STEP 6 6. Carefully apply DryStrip Cover Fluid along entire length of IPG strip.
Run IEF, STEP 7 7. Place cover on strip holder. Run IEF, STEP 8 8. Place assembled strip holder on Ettan IPGphor platform
From IEF to SDS-PAGE Choice of electrophoresis systems Amersham Biosciences 23 x 20 cm 8 x 10 cm 16 x 16 cm
Choice of electrophoresis systems Bio-Rad Protein gel staining methods
Advantage of using zinc reverse staining 1. Fast (5mins) 2. Convenient (only 2 reagents) 3. Sensitivity (1ng) 4. Compatible to mass spectrometry 5. Re-stainable by all other methods 6. Recover of protein is possible 7. Economic 8. Less toxic (require no heavy metal or organic solvent) Comparison of staining sensitivity of four methods 2008 Proteomics Lin et. al,
Find the different protein spots on 2-DE gels CNormal Patient D Prepare the protein spots for analysis by mass 1. Washing staining material from the gel 2. Equilibrium of gel to protease buffer condition 3. Reduction and alkylation of proteins 4. Digestion of proteins to peptides 5. Extraction of peptides 6. Purification of peptides (Optional)
Principle of mass spectrometry in proteomics Ion source Mass analyzer detector Ion source: ionize peptide/protein to gas ions Mass analysis: analyze ion according to mass/charge (m/z) Detection: detect the prescence of ions femtomole attomole (10-15 10-18 mole) Commonly used Mass Spectrometer in Proteomics MALDI-TOF Matrix Assisted Laser Desorption Ionization Time Of Flight ESI tandem MS (with HPLC, LC tandem MS or LC MS/MS) Electro Spray Ionization Mass Spectrometry
Commercial available MALDI-TOF Microflex, Bruker Voyager DE-PRO, ABI MALDI micro, Micromass Principle of MALDI-TOF mass spectrometry peptide mixture embedded in light absorbing chemicals (matrix) pulsed UV or IR laser (3-4 ns) detector + + vacuum + + + + + + + + V acc strong electric field cloud of protonated peptide molecules Time Of Flight tube
Typical result from MALDI-Tof (spectrum) Peptide mass fingerprinting (PMF) Gel Database tryptic digestion mass spectrometry Protein? 1 2 3 stored data or theoretical? peptides compare:?? is identical to??
ESI Quadrupole MS N 2 HPLC Gold-coated glass capillary V acc opening: 1-2 m μ pray + + + + + + + + + + + + + + + + + + + + + + + + + + + + solvents + + + + + + + + Charged droplets Taylor cones Orifice + + + Ions vacuum Quadrupole detector (multiplier) Nano electrospray: >30 min spray time for 1 μl sample Highly charged molecules are selected by ac modulation of transverse fields Principle of Quadrupole mass filter U V cos 2 π f t ions with corresponding m/z Detector U + V cos 2 π f t ions collide with the rods Scanning of measuring range: U and V are varied ac and dc voltages U (DC voltage) V (peak amplitude of a radio frequency)
Typical result from ESI Quadrupole MS From Eckerskorn in Bioanalytik, Lottspeich and Zorbas (Eds) Triple Quadrupole Mass Spectrometer Interface NH 2 ac only for proper ion entrance 1. ion scanner collision 2. ion scanner multiplier chamber Argon atoms NH 2 Quadrupole 0 Quadrupole 1 Quadrupole 2 Quadrupole 3 Detector CID: Collision Induced Dissociation for acquiring Molecular weight and Structural information
Nomenclature for CID fragments CID mass spectrum Note: Not all b or y ions will present in the spectrum
Commercial LC/MS/MS API 4000, API Q-Tof ultima API, Micromass HCT plus, Bruker Identification of protein (MASCOT) http://www.matrixscience.com/
Other proteomic techniques Applications of Proteomics 1. Protein Complexes Mining 2. Yeast Two-hybrid system (in vivo PIP) 3. Phage display and cell surface display system (in vitro PIP) 4. Protein Arrays 5. SELDI protein chips (Ciphergen) 6. Multi-dimensional HPLC (MDLC)
1. Protein Complexes Mining 1. Proteome Complex Mining A functional proteomics approach A proteome complex mining example (purification of kinases) 1. ATP is immobilized to beads in protein kinase conformation 2. Total protein is mixed the beads and the mixture washed 3. Remaining proteins isolated and identified protein kinases, and purine dependent metabolic enzymes
Proteome complex mining by affinity capture Affinity capture methods Coimmunoprecipitation (Adams et al. 2002, eg. Anti p53 antibody) Coprecipitation (Seraphin et al. 2003, eg. V5 epitope) Protein affinity-interaction chromatography (Einarson and Orlinick 2002, eg. GST fusion protein) Isolation of intact multi-protein complexes (eg. Nuclear pore complexes, ribosome complexes, and spliceosomies)
Affinity capture methods Coimmunoprecipitation Coprecipitation Protein affinity-interaction interaction Isolation of intact multi- protein complexes 2. Yeast Two-hybrid system (in vivo PIP)
2. Yeast Two-Hybrid System (in vivo) Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription. Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs. Activation Domain Prey Protein Bait Protein Binding Domain Reporter Gene Yeast 2 hybrid system, contd. X Y1, Y2, Y3, Y5, Y6 (all genome) X Y4
More complex Yeast 2/3/4 hybrid system 3. Phage display and cell surface display system (in vitro PIP)
3. Phage display system (in vitro) 3. Phage display system (in vitro) Biopanning Phage minor coat protein GIII SCIENCE VOL 298 18 OCTOBER 2002
Applications for Phage display system Human antibody techniques The growth and potential of human antiviral monoclonal antibody therapeutics Wayne A Marasco & Jianhua Sui Nature Biotechnology 25, 1421-1434 (2007) Published online: 7 December 2007
4. Protein Arrays 4. Protein (micro) arrays Another Functional Proteomics Approach Same concept as a DNA Array Has been published in a peer-reviewed journal Too much expectation lies in with.
Technological Components for Protein Chips Protein Microarrays Science, 289, 1760, 2000 Microspotting of proteins on aldehyde glass slide 150~200 µm in diameter (100 µg/ml) 10,799 spots of Protein G (1,600 spots/cm 2 ) A single spot of FRB (FKBP12-rapamycin binding)
Protein Microarray G. MacBeath and S.L. Schreiber, 2000, Science 289:1760 Spotting platform and protein microarray What protein microarray can do? 1. Protein / protein interaction 2. Enzyme / substrate interaction (transient) 3. Protein / small molecule interaction 4. Protein / lipid interaction 5. Protein / glycan interaction 6. Protein / Ab interaction Reference: 1. G. MacBeath and S.L. Schreiber, 2000, Science 289:1760 2. H.Zhu et al, 2001 Science 293:2101 3. Ziauddin J and Sabatini DM, 2001 Nature 411:107
Application of protein microarray Protein microarrays (Ab arrays)
Face the real world The true spot quality from real experiment Class of capture molecule for protein microarray
Core Technologies in Protein Chip Protein chips for practical use Detecting the the biomolecular interaction with with high high sensitivity and and reliability How to construct the monolayers of biomolecules on a solid surface Maximizing the binding efficiency Most difficult parts Maximizing the fraction of active biomolecules Minimizing the nonspecific protein binding 5. SELDI protein chips
5. SELDI protein chip SELDI surface enhanced laser desorption/ ionization Protein chips Types of protein chip IMAC30 immobilized metal affinity capture array with a nitriloacetic acid (NTA) surface with an updated hydrophobic barrier coating. IMAC3 mmobilized metal affinity capture array with a nitriloacetic acid (NTA) surface. CM10 weak cation exchange array with carboxylate functionality, with an updated hydrophobic barrier coating WCX2 weak cation exchange array with carboxylate functionality. Q10 strong anion exchange array with quaternary amine functionality, with an updated hydrophobic barrier coating. SAX2 strong anion exchange array with quaternary amine functionality. H50 bind proteins through reversed phase or hydrophobic interaction chromatography with an updated hydrophobic barrier coating H4 mimic reversed phase chromatography with C16 functionality. NP20 mimic normal phase chromatography with silicate functionality Au old chips to be used directly for MALDI-based experiments
Experimental procedure of SELDI protein chip SELDI protein chip, application Representative raw spectra and gel-view (grey-scale) of serum from a normal donor, and from patients with either BPH (benign prostate hyperplasia) or prostate cancer (PCA) using the IMAC3-Cu chip chemistry (Virginia Prostate Center).
6. Multi-dimensional HPLC (MDLC) Configuration of MDLC 2nd RP 1st SCX From: N341 Institute of Biomedical Sciences, Academia Sinica
An analysis result by MDLC From: N341 Institute of Biomedical Sciences, Academia Sinica Agilent 1100 series MDLC MASS