Genomics and Proteomics, Study notes of Genomics

Download Genomics and Proteomics and more Study notes Genomics in PDF only on Docsity! GAP- UNIT-5 The word “proteome” (term coined by Mark Wilkins in 1995) represents the complete protein pool of an organism encoded by the genome. In broader term, Proteomics, is defined as the total protein content of a cell or that of an organism. Proteomics helps in understanding of alteration in protein expression during different stages of life cycle or under stress condition. Likewise, Proteomics helps in understanding the structure and function of different proteins as well as protein-protein interactions of an organism. A minor defect in either protein structure, its function or alternation in expression pattern can be easily detected using proteomics studies. The first protein studies that can be called proteomics began with the introduction of two-dimensional gel electrophoresis of E. coli proteins (O’Ferrall, 1975) followed by mouse and guinea pig protein studies (Ksole, 1975). This is important with regards to drug development and understanding various biological processes, as proteins are the most favourable targets for various drugs. Proteomics on the whole can be divided into three kinds. Structural Proteomics: One of the main targets of proteomics investigation is to map the structure of protein complexes or the proteins present in a specific cellular organelle known as cell map or structural proteins. Structural proteomics attempt to identify all the proteins within a protein complex and characterization all protein-protein interactions. Isolation of specific protein complex by purification can simplify the proteomic analysis. Functional Proteomics: It mainly includes isolation of protein complexes or the use of protein ligands to isolate specific types of proteins. It allows selected groups of proteins to be studied its chracteristics which can provide important information about protein signalling and disease mechanism etc. Differential Proteomics: This includes determination of difference in protein expression. Techniques Involved in Proteomics Study Some of the very basic analytical techniques are used as major proteomic tools for studying the proteome of an organism. The initial step in all proteomic studies is the separation of a mixture of proteins. This can be carried out using Two-Dimensional Gel Electrophoresis technique in which proteins are first of all separated based on their individual charges in 1D. The gel is then turned 90 degrees from its initial position to separate proteins based on the difference in their size. This separation occurs in 2nd dimension hence the name 2D. The spots obtained in 2D electrophoresis are excised and further subjected to mass spectrometric analysis of each protein present in the mixture. Steps in Proteomic Analysis The following steps are involved in analysis of proteome of an organism as : 1. Purification of proteins: This step involves extraction of protein samples from whole cell, tissue or sub cellular organelles followed by purification using density gradient centrifugation, chromatographic techniques (exclusion, affinity etc.) 2. Separation of proteins: 2D gel electrophoresis is applied for separation of proteins on the basis of their isoelectric points in one dimension and molecular weight on the other. Spots are detected using fluorescent dyes or radioactive probes. 3. Identification of proteins: The separated protein spots on gel are excised and digested in gel by a protease (e.g. trypsin). The eluted peptides are identified using mass spectrometry. After first dimension run, strip gel is placed on the top of SDS-PAGE gel horizontally. The protein is already denatured in first dimension (by urea and reducing agents). Thus, the SDS used in gel running buffers sufficient to bind with already denatured protein maintain the necessary uniform negative charge for SDS-PAGE. Once the second dimension run is over, gel is separated and stained for protein visualization using methods studied in SDS-PAGE (Coomassie blue staining, Silver staining etc.) Sample Preparation of 2D PAGE Electrophoresis: The quality of 2D gels is highly dependent upon sample preparation. In general, the preparation should be as simple as possible to increase the reproducibility. Any protein modifications during sample preparation must be minimized. The three fundamental steps in sample preparation are 1. Cell disruption 2. Inactivation or removal of interfering substances 3. Solubilization of proteins Cell disruption The effectiveness of a cell lysis method determines the accessibility of intracellular proteins for extraction and solubilization. Different biological materials require different lysis strategies. • Osmotic lysis: Suspension of cells in hypotonic solution; cells swell and burst, releasing cellular contents. • Freeze-thaw lysis: Freezing of cells in liquid nitrogen and subsequent thawing. • Detergent lysis: Suspension of cells in detergent-containing solution to solubilize the cell membrane. • Enzymatic lysis: Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall (for example, cellulase and pectinase for plant cells, lyticase for yeast cells, and lysozyme for bacterial cells). • Grinding: Breaking cells of solid tissues and microorganisms with a mortar and pestle; usually, the mortar is chilled with liquid nitrogen and the tissue or cells are ground to a fine powder Inactivation or removal of interfering substances: After the cell lysis process various biomolecules are released along with proteins, like lipids, polysaccharide, nucleic acids, enzymes & etc. A number of other components are often added to disruption protocols for the inactivation or removal of interfering substance. Hypotonic buffers cause cells to burst more readily under physical shearing, and enzymes such as cellulase, pectinase, lyticase, and lysozyme are added to break down plant, yeast, and bacterial cell walls. Nucleases can be added to remove nucleic acids. Nucleic acids, particularly DNA, can interfere with IEF (for example by clogging gel pores) and increase sample viscosity. Nucleases are often employed during sample preparation, particularly with bacterial lysates. Benzonase is a nuclease with properties that make it particularly useful in sample preparation for 2-D electrophoresis. Polysaccharides Polysaccharides can interfere with electrophoresis by clogging gel pores and by forming complexes with proteins. Centrifugation may be used to remove high molecular weight polysaccharides. Phenol extraction, followed by precipitation with ammonium acetate in methanol, is a commonly used method that is very effective at removing polysaccharides in plant samples. Phenolic Compounds Phenolic compounds are found in all plants and in some microorganisms and they can modify proteins. Phenolic compounds may also be removed from the extraction by including polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) in the extraction solution. These compounds bind phenolic compounds, and the precipitated complex can be removed from the extract by centrifugation. Lipids Lipids can form insoluble complexes with proteins, but lipids can also complex with detergents, thereby reducing the detergents’ effectiveness at solublilizing protein. By adding organic solvent like phenols and chloroform lipids are separated from the sample after centrifugation. Proteases Cell disruption methods destroy the compartmentalization of a cell, causing the release of hydrolases (phosphatases, glycosidases, and proteases). These enzymes modify proteins in the lysate. So protease inhibitors are added to the lysis solution. Examples include either small molecules, such as phenylmethylsulfonyl fluoride (PMSF), aminoethyl-benzene sulphonyl fluoride (AEBSF). Solubilization of protein: Proteins in a biological sample are often associated with other proteins, integrated into membranes, or parts of large complexes. Protein solubilization is the process of breaking interactions involved in protein aggregation. which include disulfide and hydrogen bonds, van der Waals forces, and ionic and hydrophobic interactions. If these interactions are not disrupted, proteins can aggregate or precipitate, resulting in artifacts or sample loss. For successful 2-D electrophoresis, proteins must be well solubilized. Chaotropic Agents These compounds disrupt hydrogen bonds and hydrophobic interactions both between and within proteins. When used at high concentrations, chaotropic agents disrupt secondary protein structure. The neutral chaotropic agent urea is used at 5–9 M, often with up to 2 M thiourea, which can dramatically increase the number of proteins solubilized. Working principle of Mass Spectrometry. • Sample inlet: In a typical procedure, a sample, which may be solid, liquid, or gas, is convert to gaseous phase in the sample inlet chamber by the heating coil. • Ionization: Then the gaseous sample enters to the ionization chamber. A beam of electron bombards with the molecule and remove one electron from it. Now the sample M converts to M+ ion. • This also may cause some of the sample’s molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio after acceleration phase. • There are several types of ionization methods in mass spectrometry. The physical basis of ionization methods are very complex and outside the scope of the course. Most common methods are: • Electron impact ionisation (EI): Electron impact ionisation (EI) is widely used for the analysis of metabolites, pollutants and pharmaceutical compounds, for example in drug testing programmes. A stream of electrons from a heated metal filament is accelerated to 70 eV potential .Interaction with the analyte results in either loss of an electron from the substance (to produce a cation). • Matrix-assisted laser desorption/ionization (MALDI) • This method of ionization is a soft ionization method and results in minimum fragmentation of sample. This method is used for non-volatile, and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers. Sample spotted onto a metal plate and dried. Matrix plays a key role in this technique by absorbing the laser light energy and causing a small part of the target substrate to vaporize. Although, the process of forming analyte ions is unclear, it is believed that matrix which has labile protons, such as carboxylic acids, protonates neutral analyte molecules after absorbing laser light energy. • In the acceleration chamber ,Ions are accelerated so that they all have the same kinetic energy and they can move towards the mass analyser. Positive ions pass through 3 different electrodes attached to the wall. Negative electrode will accelerate the M+ ions. • In the deflection phase the ions enters to the magnetic field. Ions are deflected by a magnetic field due to differences in their masses. • The lighter the mass, the more they are deflected. It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, the more it will be deflected. • Detection: The beam of ions passing through the mass analyzer is detected by a detector on the basis of the m/e ratio. When an ion hits the metal box, the charge is neutralized by an electron jumping from the metal onto the ion. Application of Mass spectrometer. • Environmental monitoring and analysis (soil, water, and air pollutants, water quality, etc.) • Geochemistry – age determination, soil, and rock composition, oil and gas surveying • Chemical and Petrochemical industry – Quality control • Identify structures of biomolecules, such as carbohydrates, nucleic acids • Sequence biopolymers such as proteins and oligosaccharides • Determination of the molecular mass of peptides, proteins, and oligonucleotides. Identification of proteins: • The lists of the fragment masses are called the peptide mass fingerprint, which characterizes the protein. • Searching a database of fragment masses identifies the unknown sample. • The peptide masses are compared to either a database containing known protein sequences or even the genome. • This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. • They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. • The results are statistically analysed to find the best match. > Pep te Sequemang wig Moss Agee leome fore dfato . 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Alanine + Glycine + Sesine peptide - ‘Name 3-letter | 1-letter Residue code | code Mass Alanine Ala A 71.03711 Arginine Arg R_ | 15610111 Asparagine Asn N__ | 114.04293 Aspartic Acid | Asp D__ | 115.02694 Cysteine Cys c___|103.00919 Glutamic Acid Glu E 129.04259 Glutamine Gin Q 128 05858 Glycine Gly SG 57.02146 Histidine His H 137.05891 Isoleucine Te I 113.08406 Leucine Leu L 113.08406 Lysine Lys K 128.09496 Methionine Met M 13104049 Phenyalanine Phe F 147.063841 Proline Pro Pp 97.05276 Serine Ser s 87.03203 Threonine Thr T 10104768 ‘Tryptophan Trp w_| 186.07931 Tyrosine Tyr Y 163.06333 Valine Val Vv 99.06841 Relative Intensity BREESE FFI FETE a3 b3-H20 268.20 y2 232.17 286. 21 yl3 yL2 yll yl0 y9 y8_y7 y6 yS ys y3- y2 AITIMATFITOINTE(GR b2 b3 bs bs b6 bs-420 470.28 bs 488.29 vi F—rATA TM —-I--T 462.27 | |b6-H20 ylo e433 1023.83 bt bs * 41738 34 |p ys ae sey 29 yll 3 Y 7 1184.58 734.38, 12 13 yl 1267.66 1368.70 ry! 1 1 1 | i a] - 0 7m i] ~~ 1000 oh] 10 oe)