9-Proteomics-20241 9_ Proteomics and methods Zuzana Soldánová Marie Brázdová Proteomics is the large-scale study of proteins. Proteins are vital macromolecules of all living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body. 9-Proteomics-20242 Methods - determining the physical presence of cellular proteins ̶ Polyacrylamide gel electrophoresis (PAGE) ̶ SDS-PAGE ̶ Western blot ̶ Immunodetection of proteins ̶ ELISA (Enzyme-Linked ImmunoSorbent Assay) ̶ Immunoprecipitation ̶ Immunohistochemistry ̶ Isoelectric focusing ̶ Two-dimensional polyacrylamide gel electrophoresis Generally, proteins may be detected by using either antibodies (immunoassays), electrophoretic separation or mass spectrometry 9-Proteomics-20243 Protein sample collection ̶ 1st step ̶ cell or tissue suspension gain ̶ 2nd step ̶ cell or tissue breakdown ̶ by ultrasound ̶ by using a mild detergent to perforate the plasma membrane ̶ pushing cells through a small opening ̶ breaking the cells with a sealing rotary piston in a thick-walled container ̶ 3rd step ̶ obtaining a dense homogenate or extract with larger and smaller molecules from the cytosol (enzymes, ribosomes, metabolites, membrane-enclosed organelles) ̶ gain of organelles in an intact state 9-Proteomics-20244 Homogenization of cells or tissues 9-Proteomics-20245 Electrophoretic techniques the ability to move electrically charged molecules in an electric field vertical polyacrylamide gel electrophoresis (PAGE) alkaline buffers are used imparting a negative charge to the proteins - the proteins then move to the positive electrode (anode) different variants gel sandwiched between two containers filled with buffer into which the electrodes are immersed plate variant (samples are placed in wells on the upper side of the gel) 9-Proteomics-20246 Factors influencing the mobility of proteins in a gel ̶ Size ̶ with increasing molecular size, the mobility of proteins in the gel decreases (molecular sieve effect) ̶ Face ̶ globular proteins move faster than filamentous proteins ̶ Charge density ̶ charge/unit mass ̶ the higher the charge density, the higher the mobility in the gel ̶ Acrylamide concentration ̶ mobility decreases with increasing concentration 9-Proteomics-20247 Native PAGE ̶ proteins ̶ different shapes of molecules ̶ they also have different charges ̶ DNA ̶ uniform in terms of shape and charge distribution ̶ Interpretation of the electrophoretog ram in its native form is difficult. 9-Proteomics-20248 SDS-PAGE ̶ denaturing variant of PAGE ̶ commonly used method ̶ used for protein separation ̶ sample preparation ̶ dissolution in a solution containing negatively charged molecules (SDS: sodium dodecyl sulfate) ̶ elimination of disulfide bonds in proteins by a reducing agent (β- mercaptoethanol) ̶ completion of sample preparation by boiling https://ruo.mbl.co.jp/bio/e/supp ort/method/sds-page.html 9-Proteomics-20249 SDS-PAGE ̶ SDS negatively charged ̶ binds to proteins ̶ masking the protein's intrinsic charge ̶ protein denaturation and elimination of the effect of protein shape ̶ the number of SDS molecules bound to a protein is roughly proportional to its molecular weight ̶ equivalent charge density ̶ larger proteins in the gel face greater resistance ̶ Slower motion (molecular sieve effect) ̶ Proteins are separated by molecular weight only 9-Proteomics-202410 SDS-PAGE Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis. 9-Proteomics-202411 Methods of protein transmission from gel to membrane ̶ capillary transfer ̶ weight, glass plate, paper, filter paper, nitrocellulose filter, gel, filter paper, glass plates immersed in transfer buffer ̶ the dry paper absorbs the buffer by capillary forces, thus the sample is drawn from the gel to the membrane ̶ electrophoretic transfer ̶ Western transfer (proteins are negatively charged and travel to the positive electrode - anode) ̶ the driving force is the electric field strength ̶ vacuum transfer ̶ similar arrangement to capillary transfer, but the sample is pulled by vacuum ̶ faster than capillary transfer ̶ 9-Proteomics-202412 Western transfer electrophoretic transfer Western transfer (proteins are negatively charged and travel to the positive electrode - anode) the driving force is the electric field strength ̶ transfer of proteins separated by electrophoresis from a gel to a solid filter (membrane) ̶ composition: sponge, filter paper, gel, membrane, filter paper, sponge 9-Proteomics-202413 Protein visualisation - staining ̶ Non-specifically ̶ staining of all proteins in the gel ̶ Coomassie Brilliant Blue ̶ Silver ̶ Specific-Immunodetection ̶ staining of only the selected protein (membrane) ̶ antibodies Coomassie Brilliant blue Ponceau S 9-Proteomics-202414 Immunodetection of proteins 1. saturation of free binding sites on the membrane 2. cheap protein solutions (milk, BSA,...) 3. binding of the primary antibody to the respective antigen while washing the filter with a specific antibody solution 4. washing 5. binding of the secondary antibody containing the conjugated enzyme 6. washing 7. providing the appropriate substrate for the conjugated enzyme 8. signal detection 9. chemiluminescence 10. color detection 11. for example, radioactive probes or the aforementioned secondary antibodies are used to make the protein visible on the membrane Mechanism of detection chemistries. In each method of western blot detection, a detectable signal is generated following binding of an antibody specific for the protein of interest. In colorimetric detection (A), the signal is a colored precipitate. In chemiluminescence (B), the reaction itself emits light. In fluorescence detection (C), the antibody is labeled with a fluorophore. 9-Proteomics-202415 Immunodetection of proteins Patobiochemistry 9-Proteomics-202416 Antibodies ̶ they are able to recognize specific protein epitopes ̶ serve as probes ̶ distribution ̶ monoclonal ̶ they are obtained from hybridomas ̶ polyclonal ̶ they are obtained from the blood of immunized animals Immunodetection (immunological detection) is used to identify specific proteins blotted to membranes. This section provides an overview of immunodetection methods, workflow, protocol, and troubleshooting tips. •The primary antibody, which is directed against the target antigen; the antigen may be a ligand on a protein, the protein itself, a specific epitope on a protein, or a carbohydrate group •The secondary antibody, which recognizes and binds to the primary antibody; it is usually conjugated to an enzyme such as AP or HRP, and an enzyme-substrate reaction is part of the detection process (see figure below) 9-Proteomics-202417 Monoclonal antibody ̶ they are produced using hybridomas ̶ the latter arises from the fusion of a tumor leukemia cell (immortality) and Blymphocytes of an immunized animal (antibody production) ̶ expensive preparation, but in the end an almost unlimited source of antibody ̶ specific to only one epitope the fusion of a tumor leukemia cell (immortality) and Blymphocytes of an immunized animal (antibody production) 9-Proteomics-202418 Polyclonal antibodies ̶ they are obtained from the serum of immunized laboratory animals ̶ relatively cheap and quick preparation ̶ Polyclonal vs. monoclonal antibodies ̶ This summary table highlights the five main differences between the two types of antibodies. ̶ react with multiple epitopes Figure 1. A) Polyclonal antibodies bind to the same antigen, but different epitopes; and B) monoclonal antibodies bind to the same epitope on a target antigen. •Inexpensive and relatively quick to produce (+/- 3 months). •Higher overall antibody affinity against the antigen due to the recognition of multiple epitopes. •Have a high sensitivity for detecting low-quantity proteins. •High ability to capture the target protein (recommended as the capture antibody in a sandwich ELISA). •Antibody affinity results in quicker binding to the target antigen (recommended for assays that require quick capture of the protein; e.g., IP or ChIP). •Superior for use in detecting a native protein. •Easy to couple with antibody labels and rather unlikely to affect binding capability. https://www.bio-rad.com/en-cz/applications-technologies/western-blotting- immunodetection-techniques?ID=PQEEPOBWLN4A 19 9-Proteomics-202420 Primary antibody ̶ binds to a specific antigen epitope ̶ a labeled secondary antibody binds to it Primary Antibody Incubation After blocking, the membrane is incubated in a solution containing the primary antibody, usually diluted in blocking buffer. The time and temperature of incubation depends on the binding affinity of the antibody to the target protein and should be determined for each antibody individually. One hour at room temperature with gentle agitation is a good starting point. In order to reduce the background staining, the amount of Tween 20 used in the buffers is also important. Antibody Concentration The optimum antibody concentration is the dilution of antibody that still yields a strong positive signal without background or nonspecific reactions. Instructions for antibodies obtained from a manufacturer typically suggest a starting dilution range. For custom antibodies or for those where a dilution range is not suggested, good starting points are: •1:100–1:1,000 dilution when serum or tissue culture supernatants are the source of the primary antibody •1:500–1:10,000 dilution of chromatographically purified monospecific antibodies •1:1,000–1:100,000 dilution may be used when ascites fluid is the source of antibody Secondary antibodies are specific for the isotype and species of the primary antibody. For example, a goat anti-rabbit secondary is an antibody raised in goats against a primary antibody raised in rabbits. Secondary antibodies bind to a number of different conserved regions on the primary antibody, and act to amplify the signal, increasing detection sensitivity. Secondary antibodies are labelled with either an enzyme for colorimetric or chemiluminescent detection or with a fluorescent dye for fluorescent detection of the protein of interest. 9-Proteomics-202421 Secondary antibody ̶ they are specific against only a small number of primary antibodies ̶ anti-mouse IgG ̶ anti-rabbit IgG ̶ marked in different ways ̶ radioactively ̶ horseradish peroxidase (HRP) ̶ alkaline phosphatase ̶ biotin ̶ fluorescent marker 9-Proteomics-202422 Detection Methods ̶ colorimetric methods ̶ chemiluminescence ̶ bioluminescence ̶ chemifluorescence ̶ fluorescence/autoradiography ̶ gold-labeled antibodies In the past, many different methods were used for western blot detection, but now the vast majority employ enzymatic chemiluminescence or fluorescent detection. Thus, most secondary antibodies are conjugated to an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) for use with a chemiluminescent substrate or labeled with a fluorescent compound for imaging. 9-Proteomics-202423 Imunobloting 1. separation of the proteins of the given sample by gel electrophoresis 2. transfer of proteins to the membrane (western blotting) 3. incubation of the membrane with a specific antibody 4. incubating the membrane with a secondary labeled antibody 5. detection of bound antibody 9-Proteomics-202424 ELISA ̶ Enzyme-Linked ImmunoSorbent Assay ̶ uses two antibodies against one protein ̶ 2 different epitopes ̶ one antibody is bound to a carrier ̶ most often on the wall of the reaction vessel 9-Proteomics-202425 ELISA ̶ benefits ̶ high sensitivity ̶ pg/ml ̶ small amount of sample needed ̶ the possibility of using semi-automatic systems 9-Proteomics-202426 ELISA ̶ design: 1. binding of the protein to the first antibody 2. washing 3. binding of the second antibody to the protein 4. washing 5. detection of a second clot 9-Proteomics-202427 AlphaLISA ̶ modification of the ELISA method ̶ "ELISA in solution" ̶ no washing ̶ both the donor and the acceptor are bound to the target molecule ̶ the donor produces singlet oxygen ̶ the result is signal amplification ̶ a wide range of uses