2D-Gel Electrophoresis - UCV

DIDACTIC UNIT 2: MAIN TECHNIQUES Chapter 4 | Protein two-dimensional electrophoresis Professor: Mónica Díez, PhD  PROTEOME AND TECHNOLOGY Prefractionation Sample Prep...

DIDACTIC UNIT 2: MAIN TECHNIQUES Chapter 4 | Protein two-dimensional electrophoresis Professor: Mónica Díez, PhD  PROTEOME AND TECHNOLOGY Prefractionation Sample Prep Proteomic research required to separate large numbers of proteins, to identify them, and Automatization to study their modifications. Protein Separation 2-D Electrophoresis Human genome 30000 genes Study Identification Of modifications Saccharomyces cerevisiae 5885 genes Bioinformatics E.Coli 4285 genes WORKFLOW IN PROTEOMICS 3  1D vs. 2D GELS 1D-Gel 2D-Gel 4  WORKFLOW IN PROTEOMICS Ortogonality pH 3 pH 10 Mw P1 1ª dimensión high P2 2ª dimensión Improving conditions we can reach to… P2 2ª dimension Up to 4000 proteins per gel. low 5  5000 spots 2500 spots Increase of the resolution by using of gradient pH of 1 unit 6  Protein detection in 2D gels STAINING WITH CHROMOPHORES Coomassie blue. It can detect 100ng – 1mg. Silver nitrate. It can detect 5 - 10ng. STAINING WITH FLUORESCENT DYES SYPRO-ruby, SYPRO-orange, SYPRO-red, SYPRO- tangerine It can detect 1 - 10ng. RADIOACTIVE ISOTOPES Autoradiography of labeled proteins  Protein detection in 2D gels DYNAMIC RANGE of each of them Isótopos Color in which proteins are marked on the gel  Protein detection in 2D gels EXAMPLE 1: Coomassie blue Silver nitrate SYPRO Ruby  Protein detection in 2D gels ADVANTAGES INCONVENIENTS Easy to carry out It is not very sensitive Coomassie blue Economic Narrow dynamic range It is not quantitative because Very sensible different proteins tend to Economic interact with silver ions Silver nitrate Method of choice differently. Some do not even stain. It is getting better Narrow dynamic range, usually saturated. Fast SYPRO-ruby Very sensible Very expensive Wide dynamic range Very sensible Working with radioactivity Radioactive Isotopes Wide dynamic range Expensive  Protein detection in 2D gels EXAMPLE 2: One of these gels has been stained with SYPRO Ruby. Which of them? Is it possible that both gels correspond to the same sample? What has the second gel been stained with?  Detección de proteínas en las 2D EXAMPLE 2: Solution SYPRO Ruby Coomassie blue  TWO DIMENSIONAL ELECTROPHORESIS “Proteomics” is the large-scale screening of the proteins of a cell, organism or biological fluid, a process which requires stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot identification, and database searches. The core technology of proteomics is 2-DE At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure. And additionally quantify.  SAMPLE PREPARATION CELLULAR FRACTIONATION Cytosol Membranes Cardiomyocytes Improvements Higher spot number, although some appear in several gels. Information on cellular localization. Cytoskeleton Nuclei 14  AFFINITY PROTEIN FRACTIONATION EXAMPLE 15  Before runninng IEF, you should… Measure the protein conc. in your samples. – Widely used protein assay methods 1. Biuret 2. Lowry methods 3. Bradford methods 4. UV methods 5. Special methods 6. Other commercial methods. 1. BCA assay (bicinchoninic acid assay, Pierce) 2. DC protein assay (detergent compatible, Bio-rad) 3. DC/RC protein assay (detergent/reducing agent compatible, Bio-rad)  BIDIMENSIONAL ELECTROPHORESIS pH 3 pH 10 Mw alto Up to 4000 protein/gel Provides information about the protein (kDa, pI). Provides information about PTMs. Basic proteins and membranes proteins are difficult to resolve. Low abundance proteins are not detected. bajo 17 BIDIMENSIONAL ELECTROPHORESIS 18 BIDIMENSIONAL ELECTROPHORESIS 19  EVOLUTION OF 2-DE METHODOLOGY SDS-PAGE Gel size: This “O’Farrell” techniques has been used for 20 years without major modification. 20 x 20 cm have become a standard for 2-DE. Assumption: 100 bands can be resolved by 20 cm long 1-DE. Therefore, 20 x 20 cm gel can resolved 100 x 100 = 10,000 proteins, in theory. 100 100 20 2-D Proteomics Example 21  BIDIMENSIONAL ELECTROPHORESIS Two-dimensional difference gel electrophoresis 22 DIGE  SOLUBILIZATION Proteins need to be solubilized in a denaturing buffer with low ionic strength to maintain the native charges of the protein and keep them in solution. CHAOTROPIC AGENT: (7M) UREA y (2M) TIOUREA. They break down hydrogen bonds that can cause protein aggregation and formation of secondary structures that affect the mobility of the protein. (Agentes caotrópicos) NON-IONIC OR ZWITERIONIC DETERGENTS: (4%) CHAPS. They break down hydrophobic interactions and increase the solubility of proteins in their pI. They allow the migration of proteins according to their own charges. ANFOLYTES or ANFOLINES: (≤ 0.2%). They prevent the precipitation of proteins at their isoelectric point during isoelectric focusing when the salt concentration is low. (Amfolitos o anfolinas) REDUCING AGENTS : (10mM) DTT. They eliminate disulfide bridges. 23  1D: Isoelectric focusing (IEF) 1D: ISOELECTROENFOQUE (IEE) Sample Buffer contains: Chaotropes:  8M Urea  2M Thiourea/ 7M Urea Surfactants:  4% CHAPS  2 % CHAPS / 2 % SB-14 Reducing agents:  65 mM DTE (dithioerythritol)  100 mM DTT ( dithiothreitol)  2 mM tributyl phosphine Ampholytes 2% 24 First dimension, Isoelectric focusing (IEF) 25  1D: Isoelectric focusing (IEF) Proteins are separated based on their isoelectric point (pI), defined as the pH at which the net charge of the protein is zero. 26  Isoelectric Focusing The isoelectric point is the pH at which the net charge of the protein molecule is neutral. Different proteins have different isoelectric points. Isoelectric point is found by drawing the sample through a stable pH gradient. The range of the gradient determines the resolution of the separation. 27  SDS-PAGE Second Dimension. Separation by size. Run perpendicular to Isoelectric focusing. The only unresolved proteins after the first and second dimensions are those proteins with the same size and same charge – rare! 28  1D: Isoelectric focusing (IEF) 2-D – Separation is based on size and charge First step is to separate based on charge or isoelectric point, called isoelectric focusing. Then separate based on size (SDS-PAGE). 29 Narrow-Range IPG Strips pH 4 pH 5 pH 4 pH 9 pH 5 pH 6  1D: Isoelectric focusing (IEF) The IEF allows to distinguish proteins which pI differs as little as 0,01, which means that a single net charge is enough to separate them. 31  What is IEF IEF is preformed in a pH gradient. Proteins are amphoteric molecules with acidic and basic buffering groups (side chain). In basic environment, the acidic groups become negatively charged. In acidic environment, the basic groups become positively charged. The net charge of a protein is the sum of all charges. Isoelectric point (pI): the pH where the charge of a protein is zero. 32  The principle of IEF The IEF is a very high resolution separation method, and the pI of a protein can be measured. 33  1D: Isoelectric focusing (IEF) Polyacrylamide gels (3-4%) with immobilized pH gradients (IPG): Thanks to the electric current, the proteins migrate according to their charge to the point of the gel with a pH that matches their pI, and they will be focused. + - 7.1 7.1 8.8 7.1 8.4 8.8 8.4 3.9 8.8 3.9 8.4 3.7 5.3 3.7 5.3 IPG 3 10 3.7 3.9 7.1 8.4 8.8 5.3 34 2-D GELS: WORKFLOW Abnova youtube videos for 2D-Gels Protein extraction (1) http://www.youtube.com/watch?v=Z2U0_BsVXnU Protein quantification (2) http://www.youtube.com/watch?v=ZQgR9Ww9xi4 IEF sample calculation (3) http://www.youtube.com/watch?v=S_h76Vl0T6c IEF electrophoresis (4) http://www.youtube.com/watch?v=mkMPx49QZtw Gel casting (5) http://www.youtube.com/watch?v=VjrhnMRBxcI Reduction and alkylation (6) http://www.youtube.com/watch?v=SaU1qX37nPM 2D Gel Electrophoresis (7) http://www.youtube.com/watch?v=8p-zBJfJZ_0 Fluorescent Stain (8) http://www.youtube.com/watch?v=4WuUZJc5fOA 2D Gel Electrophoresis (9) Image Capture-Laser Scanner http://www.youtube.com/watch?v=Wm6svEu-H9A 2D Gel Electrophoresis (10) Visible Stain http://www.youtube.com/watch?v=atnTfPVCxH8 2D Gel Electrophoresis (11) Mass Sample Preparation http://www.youtube.com/watch?v=sz3BN7C_FSI 2D Gel Electrophoresis (12) Mass Analysis http://www.youtube.com/watch?v=G3auxOfvQbg Abnova videos http://www.abnova.com/abvideo/ 35  Two ways to form pH gradient A. Classic IEF technique Carrier ampholyte generated pH gradient. B. modern IEF technique Immobilized pH gradients 36  Problems for the traditional IEF 1. Long running time. Protein close to their pI have low net charge thus have low mobility. Denatured polypeptides migrate slower in gel than native protein. 2. Gradient drift. The pH gradient become instable during time Most basic proteins drift out of the gel. 3. Proteins behave like additional carrier ampholyte They modify the profile of pH gradient 37 B. So we use ALWAYS modern IEF technique. Immobilized pH gradients (IPG) http://www.youtube.c om/watch?v=mkMPx 49QZtw IPG strips Immobiline 13 cm pH 3-10 N/L Amersham Biosciences Bio-Rad IEF Instrument PROTEAN® i12™ IEF System — Gel-Side Up Assembly http://www.youtube.com/watch?v=nIXidk_DWpE Ettan IPGphor II Etapas: 1)5000V hasta 30000V 1h (20 oC) 2)500V o/n (20 oC) 3)5000V 30 min  2-DE instruments, 1st dimension Amersham Biosciences Bio-Rad 39  1D: Isoelectric focusing (IEF) First dimension: IEF Immobilized pH gradients (IPGs) IPG principle: pH gradient is generated by a limited number (6-8) of well defined chemicals (immobilines) which are co-polymerized with the acrylamide matrix. IPG allows the generation of pH gradients of any desired range (broad, narrow, ultra- narrow) between pH 3 and 12. sample loading capacity is much higher. This is the method of choice for micropreparative separation or spot identification. 40  1D: Isoelectric focusing (IEF) First dimension: IEF Procedure: Individual strips: 24, 18, 11, 7 cm long 3 mm wide 1. Rehydratation of 0.5 mm thickness IPGs dry strips 2. Applying the sample: in gel hydratation Cup loading 3. Running IPG strips 41  1D: Isoelectric focusing (IEF) What quantities of samples can be loaded in one IPG strip? (18 cm) Analytical run: 50-100 mg Micropreparative runs: 0.5-10 mg 42 Commercial immobilized pH gradient strips (IPG strips) Introduced by Gorg. A. Ref: Gorg. A (1994), Westermeier (2001) Dried gel strips can be stored at -20 to -80 from months to years. 43  Advantage of IPG strips 1. Industrial standard reduce variation. 2. The chemistry of the immobiline is better controllable. 3. The film-supported gel strips are easy to handle. 4. The fixed gradient are consistent during IEF. 5. Stable basic pH gradient allow reproducible results for basic proteins. 6. High protein loads are achievable. 7. Less protein loss during equilibration in buffer. 44 IPGs strips pH range 45  Solution in the IEF To maintain a gradient as stable as possible, electrode solutions are applied between the gel and the electrodes. Acid is used at the anode (+). Base is used at the cathode (-). – Example: an acidic carrier ampholyte reach the anode (+), its basic buffering group would protonated (acquire a positive charge) from the medium and it would be attracted back by the cathode. 46  Carrier ampholytes as solvents for proteins. Carrier ampholytes also help to solublize proteins, which stay in solution only in the presence of buffering compounds. They are necessary in traditional IEF and new immobilized pH gradient IEF. 47  Rehydration of IPG strip Standard rehydration solution: 8M urea, 0.5% CHAPS, 0.2% DTT, 0.5% carrier ampholyte, 10% (v/v) glycerol, 0.002% bromophenol blue Types of rehydration: 1. Rehydration cassette 2. Reswelling tray 3. Rehydration loading 4. Cup loading 48 1. Rehydration cassette Disadvantages: 1. high volume of rehydration solution needs. 2. cassette leaking due to urea and detergent. 3. rehydration loading of different sample is not possible. 49  2. Reswelling tray Rehydration volume must be controlled. 7cm: 125 mL 13 cm: 250 mL 18 cm: 340 mL 24 cm: 450 mL 50  In reswelling tray Rehydration volume is too big. Preferable reswell of LMW compounds. Leave detergent and HMW compounds outside. Over-swelling causing background smearing. Rehydration volume is too small. Pore size will be too small for HMW proteins to enter. Rehydration must perform at RT. (urea might crystalize at low temperature.) 51  3. Rehydration loading The sample in lysis buffer diluted with rehydration solution. Rehydration occurs in an individual strip holder. The dry gel matrix takes up the fluid together with the protein. Small molecules go into the gel matrix faster. Proteins diffuse into the fully hydrated gel later. It takes up to 12 hours for rehydration loading. IEF is preformed with the gel surface down. 52 IEF sample loading 53  4. Cup loading The strip is pre-rehydrated with rehydration solution. (6 hours) The sample is applied into a loading cup at a defined pH. The proteins are transported into the strip electrophoretically. IEF is preformed with the gel face up. 54 Rehydration loading v.s. cup loading Rehydration loading Cup loading 55 Comparison between rehydration and cup loading Rehydration loading Cup loading Pro Pro 1. No precipitation 1. Extreme pH condition still works 2. Less manipulation 2. Faster entry of protein, less protein- 3. Higher sample loading protein interaction 4. High entry of HMW protein 3. More protein spots developed Con 1. Protein loss for pH 6-9 or 6-11 Con 2. Rehydration time is too long 1. Protein with pI near application point 3. Protein might aggregate during tends to aggregrate rehydration 2. Does not always work well in all 4. Protein with low solubility might conditions precipitate inside the gel 56  Cover fluid Paraffin oil is widely used. Cover fluid can prevent 1. Drying of the strip 2. Crystallization of the urea 3. Uptake of O2 and CO2 Silicon oil is not recommended 57  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 3 3. Wet entire length of IPG strip in rehydration solution by placing IPG strip in strip holder (gel facing down).  Run IEF 4 4. Gently lay entire IPG strip in the strip holder, placing the end of IPG strip over cathodic electrode.  Run IEF 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 6 6. Carefully apply DryStrip Cover Fluid along entire length of IPG strip.  Run IEF 7 7. Place cover on strip holder.  Run IEF 8 8. Place assembled strip holder on Ettan™ IPGphor™ platform  Temperature Spot positions of certain proteins can vary dependent on temperature (Gorg. A. 1991) Running at 20C is optimal. Above the temp where urea might crystalize. Below the temp which cause carbamylation. Isocyanic acid reacts with amines to give ureas (carbamides): HNCO + RNH2 → RNHC(O)NH2. This reaction is called carbamylation. Active temperature control is necessary. 66 2D- Gel electrophoresis QUIZZ 67  2D- Gel electrophoresis QUIZZ 18 13 4 17 A: pI 6.4, PM 60 KDa B: pI 8.3, PM 34 KDa C: pI 6.4, PM 33 KDa D: pI 5.0, PM 100 KD 68  2D- Gel electrophoresis QUIZZ 18 13 4 17 A: pI 6.4, PM 60 KDa 13 – A 4- B B: pI 8.3, PM 34 KDa 17- C C: pI 6.4, PM 33 KDa 18- D D: pI 5.0, PM 100 KD 69  2D-PAGE Analysis Software 2D-PAGE technology has been in use for over 20 years, and potentially provides a vast amount of information about a protein sample. However, due to difficulties with data analysis, it remains only partially exploited. 70  Analysis problems It can be very difficult to compare the results of two experiments to yield a differential expression profile: Can be severe deformation of gel due to – uneven coolant flow – voltage leaks Can be problems with normalisation of – background – spot intensity Can be differences in sample preparations. 71  Current state of software Correct identification and alignment of spots from the two gels has generally been a process with a lot of manual intervention - hence very slow. The processing power available with today’s PCs means that automated analysis is starting to become possible. One vendor claims a throughput of 4 gel pairs per hour can be compared and annotated by an experienced user of their package. 72  Automated gel matching Gel matching, or “registration”, is the process of aligning two images to compensate for warp/deformation. Some packages still require the user to identify corresponding spots to help with gel matching. The Z3 program from Compugen has a fully-automated gel matching algorithm: – define set of small, unique rectangles. – compute optimal local transformations for rectangles. – Interpolate to make smooth global transformation. Note that this makes use of spot shape, streaks, smears and background structure, which other programs discard. 73 Automated gel matching 74 Automated gel matching 75  Spot detection Once the gel images have been matched, the program automatically detects spots. Algorithms are generally based on Gaussian statistics. 76  Spot Quantitation The positions of detected spots are calibrated to give a pI / mW pair for each protein. A value for the expression level of the protein can be calculated from the overall spot intensity. Some programs do not quantitate each gel separately, but calculate relative intensity pixel by pixel. This may be a more accurate approach. 77  Differential Expression The user can set threshold values for the detection of differential expression. This helps reduce the amount of information displayed at once. In this example, a protein expressed only in the second sample is circled in red. The yellow circles show proteins which are differentially expressed. 78  Annotation Some systems allow semi-automatic annotation of spots, based on a database of proteins listing their pI / mW values. Proteins of interest can also be excised from the gel and sent on to mass spectrometry for definitive identification. The ProteomeWorks system from Bio-Rad offers such an integrated solution for 2D-PAGE and MALDI. Check Bio-Rad bulletin 79  Multi-Experiment Analysis One useful feature of modern programs is the ability to collate data from many runs of the same experiment. Spots which only appear in one gel are likely to be artifacts, and are removed from the analysis. This is an excellent way to reduce noise and enhance weak signals. 80  2-DE gels analysis 2-DE software PDQUEST, MELANIE,Z3, Decyder…… 81 2-DE gels analysis 82 2-DE gels analysis 83  WORKFLOW IN PROTEOMICS “High-throughput” mapping Differential comparison Protein-interaction mapping Separation of all proteins 2-DE electrophoresis Multiprotein complexes purified by affinity (antibodies) Control Cells Apoptotic Cells Complexes separated by electrophoresis 1-D or 2-DE or 2-D nano Identification of all proteins Identification of proteins HPLC Databases 2-DE databases Bioinformatic Identification of proteins Accessible on the Net 84  Challenges for 2-DE 1. Spot number: – 10,000-150,000 gene products in a cell. – PTM makes it difficult to predict real number. – Sensitivity and dynamic range of 2-DE must be adequate. – It’s impossible to display all proteins in one single gel. 85  Challenges for 2-DE 2. Isoelectric point spectrum: – pI of proteins: range from pH 3-13. (by in vitro translated ORF) – PTM would not alter the pI outside this range. – pH gradient from 3-13 does not exist. – For proteins which pI > 11.5, they need to be handed separately. 86  Challenges for 2-DE 3. molecular weights: – Small proteins or peptides can be analysed by modifying the gel and buffer condition of SDS-PAGE. – Protein > 250 kDa do not enter 2nd SDS-PAGE properly. – 1-DE (SDS-PAGE) can be run in a lane at the side of 2-DE. 87  Challenges for 2-DE 4. hydrophobic proteins: –Some very hydrophobic proteins do not go in solution. –Some hydrophobic proteins are lost during sample preparation and IEF. –More chemical developments are required. 88  Challenges for 2-DE 5. Sensitivity of detection: – Low copy number proteins are very difficult to detect, even employing most sensitive staining methods. – Sensitivity of staining methods: 1. Silver staining 2. Fluorescent staining 3. Dye binding staining (Coomassie Blue Reagent, CBR) http://www.dnatube.com/video/3896/Protein-Quantification-CBR Compare to BRADFORD: http://www.dnatube.com/video/3953/Bradford-method-of-protein- quantification 89  Challenges for 2-DE 6. Loading capacity: – For detection of low abundant proteins, more sample needs to be loaded. – A wide dynamic range of the SDS-PAGE is required to prevent merging of highly abundant protein. – Loading capacity: IEF > SDS-PAGE 90  Challenges for 2-DE 7. Quantitation: – The detection method must give reliable quantitative information. – Silver staining does not give reliable quantitative data. 91  Challenges for 2-DE 8. Reproducibility: – Highest importance in 2-DE experiment. – Immobilized pH gradient strip have improved a lot for 1st dimension consistency – Variation most comes from sample preparation. 92  2D Gel Troubleshooting Guide Problems and solutions http://www.proteinsandproteomics.org/content/free/protocols_1/pro03.html 93 A good-looking spot pattern – streak and smear free – is not a guarantee for best 2-DE protocol. We need to be very careful 94