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Exploring Proteins Protein Separation/Purification Protein Detection and Analysis Protein Purification Required to study structure and function The challenge Cells and tissues contain a mixture of proteins(~10,000 types per cell) Proteins are diverse (size, charg...

Exploring Proteins Protein Separation/Purification Protein Detection and Analysis Protein Purification Required to study structure and function The challenge Cells and tissues contain a mixture of proteins(~10,000 types per cell) Proteins are diverse (size, charge, binding characteristics, subcellular location) Figure the best way to purify a protein  Features inform separation/purification parameters and type of assays for detection and functional analysis of protein-of-interest Jost, CBCB Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification Enrichment Protein concentration or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification Enrichment Protein concentration or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Fractionation/Enrichment of Proteins by Centrifugation Separates heterogeneous particles in suspension by gravity not one solution/mixed properties Suitable for molecules in native state, including complexes final active properly folded/functional form of protein Rate of sedimentation determined by: – Mass of particle – Density of particle (weight/volume) – Medium/solution in which particle is suspended in Preparative applications Analytical applications Separate/fractionate/resolve Measure physical properties of large quantities of molecules (typically small quantities of) macromolecules (size, shape, density) Jost, CBCB Cellular Subfractionation for Isolation of Intracellular Proteins Cell lysis – Osmotic shock (hypotonic lysis) – Sonication – Grinding up in blender – Forcing through small orifice – Detergent so when we lyse the cell it releases proteases in the soltution and we dont want these proteases in our solution so we have to inhibit them Protease inhibition by low temperature or protease inhibitors inhibitors Fractionation of cellular components from homogenate by differential centrifugation cell is broken down into its components by centrifugation Recovery of enriched fraction – Collection of pellets and supernatants Svedberg Constant (S) Derived from sedimentation coefficient s 𝑺𝑺 = 𝟏𝟏𝟏𝟏−𝟏𝟏𝟏𝟏 𝐬𝐬 – The smaller s, the slower a particle moves in a centrifugal field – Affected by particle mass of particle – Affected by shape and density of particle (friction) – Affected by medium density Quantifies rate of movement of a particle through a medium when subjected to centrifugal force – ~1-10S for proteins – 70S for prokaryotic ribosomes why does a smaller particle move slower in a centrifuge? – 80S for large eukaryotic ribosomes Jost, CBCB Sedimentation of Cellular Components (soluble) proteins differ in mass but little in density explain Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Enrichment of Cellular Compartments by Differential Centrifugation diff centri = helps you eliminate to get to molecules you want ultracentrifugation at this high speed = 100,000g nuclei at the bottom Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Sedimentation Velocity Centrifugation spin your particles through a gradient; often used with sucrose Rate-zonal or band centrifugation (High-speed) Centrifugation of particles through a solution of increasing density until sufficient separation is reached – Shallow gradient, e.g., 5-20% sucrose what does this mean , has less resolution Preparatory technique – Separates by mass and shape, such as organelles, macromolecular complexes, globular proteins Jost, CBCB Rate-zonal Centrifugation more dense?? capsid intact virus Jost, CBCB Sedimentation Equilibrium Centrifugation Analytical technique to analyze particles – e.g., mass of native proteins, protein or supramolecular complexes Separates by buoyant density through steep gradient by (lower-speed) centrifugation until equilibrium is reached between sedimentation and diffusion Extremely sensitive – High-resolution separation of similar macromolecules – e.g., DNA molecules of equal lengths composed of different isotopes Has better resolution Alberts, B. (2015). Molecular biology of the cell. New York, NY, Garland Science, Taylor and Francis Group. Jost, CBCB Equilibrium Density-gradient Centrifugation Jost, CBCB Salt Fractionation of Proteins aka “Salting out” proteins ammonium sulfate interferes with solubility of proteins Precipitation of proteins by salt – Disrupts solubility of some proteins by interfering with charges, hydration, and solute how does this happen? – Salt concentration required for precipitation varies among proteins, allowing fractionation from bulk purification by sequential concentration increase (e.g., 0.8 M for fibrinogen, 2.4 M for serum albumin) Ammonium sulfate (NH4)2SO4 commonly used since highly soluble and not harmful to proteins Also used to concentrate dilute protein solutions Requires subsequent salt removal by dialysis or gel filtration chromatography Jost, CBCB Dialysis red particles = proteins Salt removal by equilibrating protein solution with suitable buffer Protein solution in semi- permeable membrane with defined pore size – Permeable for small molecules and ions, but not proteins Incubated until equilibrium of small molecules or salts between inside and outside of membrane is reached Repeated buffer replacement to increase salt removal Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification Enrichment Protein concentration or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Liquid Chromatography Separates molecules in mixture by partitioning between mobile (liquid) and stationary phase (matrix) depending on their interaction with the matrix Separation parameters in main chromatography types: – Size (Gel filtration/size-exclusion chromatography) – Charge (Ion exchange chromatography) – Binding affinity (Affinity chromatography) often you need to use two or three methods or just try out which method will work better Lehninger, A. L., D. L. Nelson and M. M. Cox (2013). Lehninger principles of biochemistry. New York, W.H. Freeman. Jost, CBCB big molecules diffuse first orange ones because they do Larger proteins not fit in the pores , green will stick to the pores b/c they fit elute first Ion-Exchange Chromatography Separates molecules based on net charge – Affected by composition, pH, ionic strength Column containing beads with covalently attached charged groups Same charge molecules will – Anion exchangers with cationic groups – Cation exchangers with anionic groups Elution of bound molecules by increasing salt concentration in order to release beads we need to salt it out Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB  Bound proteins eluted by salt Proteins with same charge as column beads (or neutral) elute first Affinity Chromatography Separates based on specific binding affinities between molecules or groups – Antibodies to specific antigen – Transcription factors to specific DNA sequence – Enzymes to specific substrate Column with beads coated with specific binders – Retention of ligands in column Elution of bound molecules by adding excess binding partner or salt or pH change Jost, CBCB any antibodies bound will connect to the beads Bound proteins eluted by salt or pH change Weak binders elute first Elution of Bound Proteins by Excess Ligand glucose added to elute excess bound Elution of bound proteins by excess ligand Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB High Performance Liquid Chromatography (HPLC) aka High Pressure LC Increases interaction surface by using fine and dispersed column material Requires high pressure to push samples through Very high resolution and rapid separation Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification Enrichment Protein concentration or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Gel Electrophoresis (mostly) Analytical technique Separation of molecules in electrical field through porous matrix acting as molecular sieve – Polyacrylamide – Agarose Migration velocity depends on mass, charge, shape of particle and medium properties (viscosity) Native or denaturing Jost, CBCB Polyacrylamide Gel Electrophoresis (PAGE) Polymerization product of acrylamide and bisacrylamide – Creates mesh – Pore size determined by concentration and ratio of reactants Gels cast into thin slabs Vertical electrophoresis Berg, J. M., J. L. Tymoczko, G. J. Gatto and L. Stryer (2019). Biochemistry. New York, W.H. Freeman/Macmillan Learning. Jost, CBCB SDS-PAGE For denaturing PAGE what would sds denature a protein down to Amphipathic detergent Denatures – Solubilizes proteins which ones- hydrophob inter/bonds, ionic,van der walls – Disrupts non-covalent interactions → Dissociates multimeric proteins protein with a lot of subunits Reduces → Denatures proteins by binding to hydrophobic disulfide bonds residues – Masks intrinsic charge of proteins Binds at ~1 SDS/2 amino acids charge is proportional to the length , sds binds at constant ratio confers negative net charge, similar charge-to-mass ratios and similar shapes Often combined with reducing disulfide bonds Hydrophobic tail interacts with the protein’s hydrophobic regions, helping unfold and denature the protein. Hydrophilic head gives a uniform negative charge to the protein, making separation based solely on size possible. Alberts, B. (2015). Molecular biology of the cell. New York, NY, Garland Science, Taylor and Francis Group. Jost, CBCB Electrophoretic Mobility Relative mobility inversely proportional to log of mass (denaturing PAGE) – Linear relationship (most proteins) – Deviations due to modification (e.g., extensive glycosylation), extensive membrane-spanning portions, etc. Useful to estimate molecular mass – Compared to standards of known mass Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Differenceb/ t running a complex with or without dentauring can see if proteins are bonded by disulfid bonds Alberts, B. (2015). Molecular biology of the cell. New York, NY, Garland Science, Taylor and Francis Group.  homodimer -S-S- -S-S- reducing sds-page non-reducing SDS SDS SDS 2-ME/DTT ± 2-ME/DTT 2x 2x -S-S- 1x -SH two proteins but have the same size break a part the bond single band higher b/c it running with two singe bands  heterodimer -S-S- -S-S- SDS/Urea SDS/Urea SDS/Urea 2-ME/DTT ± 2-ME/DTT 1x different sizes /different bonds 1x -SH -S-S- 1x 1x 1x -SH smaller one is hgher band, mass will be approximately the two bands Detection of Proteins in SDS-PAGE Gels Protein dyes with different sensitivity of detection Coomassie Blue – Organic dye what size proteins can we use wotj SDS PAGE – Visible light Silver staining – Visible light Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Fluorescent dyes (e.g., SYPRO orange) – Requires excitation with appropriate wavelength and filters for capturing emission https://www.gbiosciences.com/Protein-Research/FASTsilver Clinical Correlation: Plasma Protein (non-denaturing) Electrophoresis non reducing EPhoresis, migrate based on mass and charge. Ran at an alkaline ph (8-9) Non-denaturing electrophoresis at alkaline pH (>most plasma protein pI) Diagnosis of certain hematological diseases based on levels of plasma proteins Devlin, T. M. (2011). Textbook of biochemistry : with clinical correlations. Hoboken, NJ, John Wiley & Sons. Jost, CBCB Isoelectric Focusing (IEF) Analytical method based on differences in isoelectric point (pI) use differences in PI as a seperation technique – pH at which net charge of molecule = 0 – Depends on content of acidic and basic groups and post-translational modification Separates proteins through pH gradient based on intrinsic charge – Formed by electrophoresis of polyampholyte mix once the proteins move to their PI it becomes neutral – Protein migration stops at pH corresponding PI positive charge will move to the right negative charge will move to the left stops migrating when its at PI proteins are sorted by their PI https://www.bio-rad.com/en-us/applications-technologies/isoelectric-focusing-2-d- electrophoresis?ID=LUSQLK2B7 Jost, CBCB Isoelectric Focusing (IEF) Alberts, B. (2015). Molecular biology of the cell. New York, NY, Garland Science, Taylor and Francis Group. Jost, CBCB Two-Dimensional (2D) Electrophoresis Combines SDS-PAGE with isoelectric focusing For high-resolution separation of protein mixtures 1st dimension: IEF in single-lane gel Electrophoresis without SDS Separation based on charge 2nd dimension: SDS-PAGE Have to do it in order, because if we do SDS page first, this will make the charge negative because it the sds sticks to the molecule and we will have nothing to seperate after Perpendicular to 1st dimension Separation based on mass Jost, CBCB PROTEOME Protein Purification Assessment * the > specific activity the more purer your protein *1u (IU): amount of enzyme required to convert 1µmol substrate/min (25°C, optimal conditions) Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification DONT NEED Enrichment Protein concentration TO KNOW or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Protein Purification Workflow Identify Source Monitoring Purification Gel electrophoresis Fractionation/ Quantification Enrichment Protein concentration or (enzyme) activity Purification Analysis Structure, function Jost, CBCB Specific Protein Detection Recognition by specific binder – Typically specific antibody using immunological methods – Ability to bind particular ligand Catalysis of particular reaction (enzyme) enzymatic Activity: Another way to detect specific proteins, particularly enzymes, is by monitoring their catalytic activity. Enzymes catalyze Ability to Bind a Particular Ligand: Some proteins may be specific biochemical reactions, and detecting these reactions (such as detected by their ability to bind specific ligands (small color changes in substrate products) can help identify and analyze the molecules or other proteins). For example, proteins that enzyme responsible. interact with DNA or other molecules can be identified using This approach is useful when studying the functional properties of their natural binding partners as probes. enzymes in the context of protein structure-function relationships. Specific Antibody Binding: One of the most common methods for detecting proteins is using specific antibodies. Antibodies are highly selective molecules that can bind to particular proteins (antigens) with great precision. Ability to Bind a Particular Ligand: Some proteins may be detected by their ability to bind specific ligands (small molecules or other proteins). For example, proteins that interact with DNA or other molecules can be identified using their natural binding partners as probes. Jost, CBCB Antibodies (Immunoglobulins) antibodies used in affinity chromatography or Proteins produced in analytical technique response to (foreign) substances (antigens) – Produced by B-lymphocytes – Bind antigens with high Light chain specificity and high affinity ~25kD Analytical application for specific protein detection ~ 50kD Preparatory application for affinity chromatography different binding site to bind different proteins purification of proteins why are anitbodies good for affinity chromatopgraphy, how do they help us analyze protein structure and function Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB subtrate enzyme,anitbody?? Mono- and Polyclonal Antibodies Monoclonal Antibodies Polyclonal Antibodies (MoAbs) (PAbs) Derived from single B-cell clone Mixture of antibodies, pooled All Abs have the same defined from several different clones specificity and recognize the Recognize same antigen, but same antigenic determinant different antigenic determinants (epitope) (epitopes) Clone #2 2 B2 Clone #1 Clone #3 B1 1 Ag 3 B3 Clone #4 4 B4 Jost, CBCB Western (Immuno-) blotting Detects antigens (proteins) after separation of proteins and transfer onto a matrix using antibodies Protein expression levels across tissues or treatment conditions Protein modification (e.g., phosphorylation using phosphoprotein-specific antibodies) Protein degradation and cleavage products Protein-protein interaction (in combination with co-immunoprecipitation) Jost, CBCB Immunoblotting (Western Blotting) Steps While secondary antibodies can be either monoclonal or polyclonal, polyclonal secondary antibodies are more commonly used because they provide stronger signal amplification and broader epitope recognition. They help better bind and amplify the 1st monoclonal antibody. 5. This will tell you the position the first antibody can be mono or poly primary antibody bound on the protein matrix. antibody recognizes Overview of Western Blotting: Proteins are separated based on size using SDS-PAGE. Proteins are transferred to a membrane (nitrocellulose or PVDF). The membrane is blocked to prevent non- specific binding. makw sure to block The target protein is detected using a othe rproteins primary antibody (monoclonal or before adding polyclonal) that binds to the protein of specific antibody interest. mono/polyclonal antibodies can help us detect the primary antibody. A secondary antibody is applied, which 1. seperate using sds-page or something else binds to the primary antibody and usually 2.transsfer your proteins to your membrane (can use electrical gradient bc the has a detection marker (e.g., enzyme or membrane will still have SDS on it ( which has a negative charge) fluorescent tag). 3. then you incubate the filter with an (binder) antibody, the protein recogonizes the Signal detection (via chemiluminescence, antibody(2). You cant visualize it yet colorimetric, or fluorescence) allows 4. Apply a seconary antibody that recognizes the first antibody ( doesn't bind to any visualization of the protein. other proteins). The secondary antibodu has an enzyme-lined with it, if you add substrate for the enzyme which leads to color productioon (fluorescence Jost, CBCB Western/Immunoblotting Examples (Image source: Shen et al, Cancer Res. 2004;64(24):9018-9026. doi:10.1158/0008-5472.CAN- 04-3262 interpret western blot https://www.cellsignal.com/products/9663/ applications?index=1&application=all Jost, CBCB Enzyme-Linked Immunosorbent Assay (ELISA) antibody Quantification of proteins (and other analytes) in fluids – e.g., antibodies or viruses in serum, drugs in urine, etc. Uses enzymes bound to specific antibodies – HRP (horseradish peroxidase) – AP (Alkaline phosphatase) Quantitation of analyte by enzyme reaction in comparison to standard – Chromogenic, luminescent or fluorescent detection Jost, CBCB ELISA Formats e.g., Detection and quantification of patient serum antibodies recognized by antivody if antibody is present it will bind to antigen e.g., Detection and quantification of viral particles in body fluids Berg, J. M., J. L. Tymoczko and L. Stryer (2007). Biochemistry. New York, W.H. Freeman. Jost, CBCB Immunofluorescence Microscopy Detection and localization of proteins within cells or tissues – Specific antibodies conjugated with fluorescent dyes – May be combined with other fluorescent dyes Detection of relocation of proteins between cellular compartments Visualization by fluorescence microscopy Jost, CBCB Immunolocalization of Antigens by Immunofluorescence goes from being concentrated receptor is more concentrated in in the cytoplasm to the nucleus the nucleis b/c Jost, CBCB 3/17/11 4:52 PM