Protein Separation and Identification Techniques PDF

Protein Separation and Identification Techniques Ana Peixoto Gomes 2024/2025 Protein Separation and Identification Techniques Proteome complexity Post-translational Genome...

Protein Separation and Identification Techniques Ana Peixoto Gomes 2024/2025 Protein Separation and Identification Techniques Proteome complexity Post-translational Genome Alternative promoters Transcriptome modifications (PTM) Proteome ~20-25 000 genes Alternative splicing mRNA editing ~ 100 000 transcripts > 1 000 000 proteins Protein Separation and Identification Techniques Post-translational modifications (PTM) May occur: After translation, proteins need assistance to fold correctly or be guided to their proper location within the cell; After folding, in their celular stations –switch on/off catalytic activity Protein Separation and Identification Techniques Proteome complexity More than 300 modification forms known: Single protein may carry several modifications Modified proteins show different properties compared to unmodified counterparts In most cases, we do not know the origin or the biological significance of the observed heterogeneities Proteomics is the study of the proteome – investigating how different proteins interact with each other and the roles they play within the organism. Protein expression can be inferred by studying the expression of mRNA, which is the middle man between genes and proteins, mRNA expression levels do not always correlate well with protein expression levels mRNA does not consider protein post- translational modifications, cleavage, complex formation and localization, or the many variant mRNA transcripts that can be produced; all of which are key to protein function.  What are the key questions that proteomics can answer? Proteomic research provides a global view of the processes underlying healthy and diseased cellular processes at the protein level. Beynon RJ. The dynamics of the proteome: strategies for measuring protein turnover on a proteome-wide scale. Brief Funct Genomic Proteomic. 2005;3(4):382-390. doi: 10.1093/bfgp/3.4.382. Garrels JI. Proteome. In: Brenner S, Miller JH, eds. Encyclopaedia of Genetics. London: Academic Press; 2001:15 What are the key questions that proteomics can answer? Which proteins are normally expressed in a particular cell type, tissue or organism as a Protein identification whole, or which proteins are differentially expressed? Measures total (“steady-state”) protein abundance, as well as investigating the rate of Protein quantification protein turnover (i.e., how quickly proteins cycle between being produced and undergoing degradation). Where a protein is expressed and/or accumulates is just as crucial to protein function as Protein localization the timing of expression, as cellular localization controls which molecular interaction partners and targets are available. Post-translational modifications can affect protein activation, localization, stability, Post-translational interactions and signal transduction among other protein characteristics, thereby adding modifications a significant layer of biological complexity. This area of proteomics is focused on identifying the biological functions of specific Functional proteomics individual proteins, classes of proteins (e.g., kinases) or whole protein interaction networks. Structural studies yield important insights into protein function, the “druggability” of Structural proteomics protein targets for drug discovery, and drug design. Investigates how proteins interact with each other, which proteins interact, and when Protein-protein interactions and where they interact. HOW???? Low-throughput methods Chromatography-based methods Gel-based methods Antibody-based methods Proteomics Techniques High-throughput methods Mass spectrometry-based proteomics Proteomics Techniques How does it start? Proteomics workflow - SAMPLE - EXTRACTION - SEPARATION - DETECTION - IDENTIFICATION - FUNTIONAL ANALYSIS, STRUCTURE,… Protein extraction The goal of this step is to break open cells to release their contents, including the target protein. It can be achieved by different approaches, such as mechanical disruption (commonly used for bacterial and yeast cells), chemical disruption (using detergents, organic solvents or chaotropic agents) and enzyme disruption (which can help break down cell walls, especially in bacterial cells). Freeze-thaw cycles and pressure cycling are alternative methods to lyse cells effectively. Protein extraction - Non-mechanical methods Detergents: Solubilize cell membranes by disrupting lipid bilayers. Some common detergents include Triton X-100 and SDS. This process is easy to use and very effective for membrane proteins, but it can cause denaturation if used at high concentrations. It may require removal before further purification. Organic solvents: Solvents such as ethanol or acetone are used to precipitate proteins and disrupt membranes. It is a quick approach, but it is not suitable for all protein types, as it can cause denaturation. Chaotropic agents: Disrupt the hydrogen bonding network in proteins, aiding in solubilization. Examples include urea and guanidine hydrochloride. This approach is especially useful for insoluble proteins but can denature proteins and often requires subsequent refolding steps. Freeze-thawing: This procedure involves repeated cycles of freezing and thawing to lyse cells by ice crystal formation. It is a simple way to lyse cells since it doesn’t require special equipment. However, it is time-consuming and not very efficient for some cell types. Enzymatic treatment: Sometimes enzymes, such as lysozymes, are used to break down bacterial cell walls, in combination with other methods for enhanced efficiency. This approach is very specific and maintains protein integrity. Unfortunately, its use is limited to bacterial cells and requires additional steps for a complete cell lysis. Protein purification techniques - SEPARATION Protein purification involves several techniques, each tailored to isolate proteins based on specific properties like size, charge or affinity. Centrifugation Separates components based on their density by spinning samples at high speeds. During the process, heavier particles, such as cellular debris, sediment at the bottom, allowing the lighter supernatant, which contains the proteins, to be collected. This technique is often the first step in purification, used to remove large contaminants and concentrate proteins from crude extracts Protein purification techniques - SEPARATION Precipitation Involves altering the solubility of proteins to cause them to aggregate and precipitate out of solution. This can be achieved using salts (salting out), such as ammonium sulphate, organic solvents or changes in pH. Although this process can lead to the co-precipitation of contaminants, it is useful for the initial protein concentration and fractionation, particularly when working with large volumes Protein purification techniques - SEPARATION Chromatography Is one of the most significant bioanalytical techniques used in different branches of life sciences and chemistry. It allows the separation, identification, and purification of the compounds of diverse origin, class, and nature from a complex mixture qualitatively and quantitatively. The separation of a compound with desired shape, size, charge, and groups can be carried out based on its capacity in terms of its binding specificity. Protein purification techniques - SEPARATION Proteins Chromatography comprises of a varied set of versatile and widely used purification techniques that can be used to separate proteins based on various properties  Protein purification techniques - SEPARATION Affinity Chromatography The stationary phase consists of a support medium, on which the substrate (ligand) is bound covalently, in such a way that the reactive groups that are essential for binding of the target molecule are exposed. As the crude mixture of the substances is passed through the chromatography column, substances with binding site for the immobilized substrate bind to the stationary phase, while all other substances is eluted in the void volume of the column. Once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength or polarity conditions. Protein purification techniques - SEPARATION Ion exchange chromatography – Separates proteins based on their charge Proteins bind to charged resins (cationic or anionic) and are eluted by increasing the ionic strength or changing the pH of the buffer. This is useful for proteins with well-defined charge, and it is often used as an intermediate purification step ❖ Cationic exchangers possess negatively charged group, and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials, because their negative charges result from the ionization of acidic group. ❖ Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called “Basic ion exchange” materials. Protein purification techniques - SEPARATION Size exclusion chromatography (SEC) ❑ Separates proteins based on size by passing them through a column filled with porous beads. ❑ Smaller proteins enter the pores and elute later, while larger proteins bypass the pores and elute earlier. ❑ This technique is usually used for separating monomers from aggregates and as a final polishing of the protein purification process. Protein purification techniques - SEPARATION Hydrophobic interaction chromatography (HIC) Separates proteins based on hydrophobicity. Proteins bind to hydrophobic groups on a resin at high salt concentrations and elute as the salt concentration decreases. This approach is appropriate for proteins containing highly hydrophobic domains and helps remove undesired aggregates. Protein purification techniques - SEPARATION Ultrafiltration and dialysis Dialysis is a process that involves placing the Ultrafiltration is a methodology that uses protein solution inside a membrane with semipermeable membranes to concentrate and selective permeability, allowing small molecules desalt protein solutions by applying pressure. to diffuse out to a surrounding solution. These methodologies are used to remove small contaminants, exchange buffers and concentrate proteins. Protein purification techniques - SEPARATION Electrophoresis of Proteins – Gel-based methods Refers to the movement of charged molecules in response to an electric field, resulting in their separation In an electric field, proteins move toward the electrode of opposite charge. The rate at which they move (migration rate, in cm2/Vsec) is governed by a complex relationship between the physical characterizes of both the electrophoresis system and the proteins. Factors affecting protein electrophoresis include the strength of the electric field, the temperature of the system, the pH, ion type, and concentration of the buffer as wall as the size, shape and charge of the proteins. These gel-based methods are used to separate proteins before further analysis by e.g., mass spectrometry (MS), as well as for relative expression profiling. Protein purification techniques Protein Electrophoresis Workflow Protein purification techniques Electrophoresis Gel Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS - PAGE) SDS and polyacrylamide remove the impact of protein structure and charge. It separates the protein based on the length of the polypeptide chain - Smaller proteins migrate a further distance through gel pores Why is SDS added to Polyacrylamide gel? The SDS breaks the disulfide bonds and modifies the protein structure. These modified structures and increased negative charge help to maintain the charge-to-mass ratio in electrophoresis. What is Polyacrylamide? Polyacrylamide is a synthetic linear polymer which is composed of acrylamide or a mixture of acrylamide and acrylic acid. It is water-soluble and used in pulp and paper industries. Protein purification techniques Native Polyacrylamide Gel Electrophoresis (NATIVE PAGE) - does not require denaturing chemicals in the gel matrix Protein purification techniques Electrophoresis Gel Two-dimensional gel electrophoresis (2DE or 2D-PAGE), Electric current to separate proteins in a gel based on their charge (1st dimension) - Isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI). Second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) - separates proteins according to their molecular weights (MW). Advantages: –A powerful technique for simultaneous separation of thousands of proteins –Relative easy to handle and affordable –High-sensitivity visualization of proteins Protein purification techniques Electrophoresis Gel Two-dimensional gel electrophoresis (2DE or 2D-PAGE), Protein purification techniques Electrophoresis Gel Differential gel electrophoresis (DIGE) is a modified form of 2DE that uses different fluorescent dyes to allow the simultaneous comparison of two to three protein samples on the same gel. Blue native PAGE (BN-PAGE) can be used for one- step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Electrophoresis Detection Electrophoresis Detection Coomassie Blue Staining is a quick, simple, and affordable method for detecting proteins on gels. It has a detection limit of 0.1–0.5 mg/protein, sensitive enough for most daily needs. During the complex formation of Coomassie Brilliant Blue with the protein, the reaction that occurs includes the following steps: The dye transfers a free electron to the groups of the proteins that can be ionized. The transfer of electrons to protein disrupts its native structure and exposes its hydrophobic pockets. The non-polar components of the dye bind to the hydrophobic pocket of the protein through van der Waals forces. The ionic interaction and complex formation through the reaction allow the visualization of the protein bands separated in protein gel Electrophoresis Detection Western-Blot or immunoblotting: widely used laboratory method for detecting and identifying specific proteins in a complex mixture. It allows researchers to determine the presence, quantity, and molecular weight of a target protein within a sample. 1. Sample Preparation 2. SDS-PAGE Gel Electrophoresis 3. Protein Transfer 4. Blocking 5. Primary Antibody Incubation 6. Secondary Antibody and Protein Detection 7. Image Acquisition and Analysis Problem-Solving You are tasked with purifying a protein from a complex mixture. The protein has a molecular weight of 50 kDa and a pI of 6.2. Design a purification protocol using two different techniques and justify your choices.