PROTEOMICS INTRODUCTION AND TECHNIQUES
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This document provides an overview of proteomics, including its definition, types, and analysis steps. It discusses several techniques used in proteomics, such as chromatography, ELISA, Western blotting, protein microarrays, Edman sequencing, SDS-PAGE, 2D gel electrophoresis, 2D-DIGE, SILAC, iTRAQ, X-ray crystallography, and mass spectrometry. The techniques allow for studying protein expression, structure, functions, interactions, and modifications to better understand cellular processes.
PROTEOMICS INTRODUCTION AND TECHNIQUES
- 1. Group # 3 Summary Proteomics 1. Introduction Berzelius in 1838 given the title “protein”, which is originated from the Greek word, proteios, meaning “the first rank” (1). The “proteome” can be defined as the overall protein content of a cell that is characterized with regard to their localization, interactions, post- translational modifications and turnover, at a particular time. The term “proteomics” was first used by Marc Wilkins in 1996 to denote the “PROTein complement of a genOME”. Proteomics is crucial for early disease diagnosis, prognosis and to monitor the disease development. Furthermore, it also has a vital role in drug development as target molecules. Proteomics is the characterization of proteome, including expression, structure, functions, interactions and modifications of proteins at any stage. The proteome also fluctuates from time to time, cell to cell and in response to external stimuli. 2. Types of proteomics I. Expression proteomics: Expression proteomics is used to study the qualitative and quantitative expression of total proteins under two different conditions. Like the normal cell and treated or diseased cell can be compared to understand the protein that is responsible for the stress or diseased state or the protein that is expressed due to disease. Compare tumor tissue sample and the normal tissue can be analyzed for differential protein expression. 2-D gel electrophoresis, mass spectrometry technique were used to observed the protein expressional changes. II. Structural proteomics:Structural proteomics helps to understand three dimensional shape and structural complexities of functional proteins. Structural proteomics can give
- 2. detailed information about the structure and function of protein complexes present in a specific cellular organelle. It is possible to identify all the proteins present in a complex system such as membranes, ribosomes, and cell organelles and to characterise all the protein interactions that can be possible between these proteins and protein complexes. Different technologies such as X-ray crystallography and NMR spectroscopy were mainly used for structure determination. III. Functional proteomics: Functional proteomics explains understanding the protein functions as well as unrevealing molecular mechanisms within the cell then depend on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact, be strongly suggestive of its biological function. Furthermore detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein- protein interactions in-vivo. 3. Steps in proteomic analysis: The following steps are involved in analysis of proteome of an organism. 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. 4. Techniques 4.1. Conventional techniques 4.1.1. Chromatography based techniques
- 3. 4.1.2. Enzyme-linked immunosorbent assay: The ELISA is highly sensitive immunoassay and widely used for diagnostic purpose. The assay utilizes the antigen or antibodies on the solid surface and addition of enzyme-conjugated antibodies to and measure the fluctuations in enzyme activities that are proportional to antibody and antigen concentration in the biological specimen. Wheat proteins causes allergic reactions in susceptible individuals that have been traced in foods to protect wheat-sensitive individuals using commercially available ELISA kits. 4.1.3. Western blotting: Western blotting is an important and powerful technique for detection of low abundance proteins that involve the separation of proteins using electrophoresis, transfer onto nitrocellulose membrane and the precise detection of a target protein by enzyme-conjugated antibodies. Western blotting is a dominant tool for antigen detection from various microorganisms and is quite helpful in diagnosis of infectious diseases. 4.2. Advanced techniques 4.2.1. Protein microarray: Protein microarrays also known as protein chips are the emerging class of proteomics techniques capable of high-throughput detection from small amount of sample. Protein microarrays can be classified into three categories; analytical protein microarray, functional protein microarray and reverse-phase protein microarray. 4.2.1.1. Analytical protein microarray: Antibody microarray is the most representative class of analytical protein microarray. After antibody capture, proteins are detected by direct protein labeling. These are typically used to measure the expression level and binding affinities of proteins. High throughput proteome analysis of cancer cells was carried out through antibody microarray for differential protein expression in tissues derived from squamous carcinoma cells of oral cavity. Antibody array was also used for protein profiling of bladder cancer. Analytical and experimental approaches have been developed for identification of cellular signaling pathways and to characterize the plant kinases through protein microarray. 4.2.1.2. Functional protein microarray: Functional protein microarray is constructed by means of purified protein, thus permits the study of various interactions including protein–DNA, protein–RNA and protein–protein, protein–drug, protein– lipid,
- 4. enzyme–substrate relationship. The first use of functional protein microarray was to analyze the substrate specificity of protein kinases in yeast . Functional protein microarray characterized the functions of thousands of proteins. The protein–protein interaction of A. thaliana was studied and Calmodulin-like proteins (CML) and substrates of Calmodulin (CaM) were identified. 4.2.1.3. Reverse-phase protein microarray: Cell lysates obtained from different cell states are arrayed on nitrocellulose slide that are probed with antibodies against target proteins. Afterwards, antibodies are detected with fluorescent. For protein quantification, reference peptides are printed on slides. These microarrays are used to determine the altered protein indicative of a certain disease. Reverse-phase protein microarray approach was evaluated for quantitative analysis of phosphoproteins and other cancer-related proteins in non-small cell lung cancer (NSCLC) cell lines by monitoring the apoptosis, DNA damage, cell-cycle control and signaling pathways. 4.2.2. Edman sequencing: Edman sequencing was developed by Pehr Edman in 1950 to determine the amino-acid sequence in peptides or proteins. The method comprises chemical reactions that eliminate and identify amino acids residue that is present at the N-terminus of polypeptide chain. The proteins from leaf sheaths of rice were extracted and analyzed through MS and Edman sequencing to determine its function. The amino-acid sequence of majority of proteins analyzed by both techniques have similar results, therefore suggesting the use of these techniques in combination for the identification of plant protein. Gel-based approaches Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis: SDS-PAGE is a high resolving technique for the separation of proteins according to their size, thus facilitates the approximation of molecular weight. Proteins are capable of moving with electric field in a medium having a pH dissimilar from their isoelectric point. Different proteins in mixture migrate with different velocities according to the ratio between its charge and mass. However, addition of sodium dodecyl sulfate denatures the proteins, therefore separate them absolutely according to molecular weight. The antigenic proteins of Streptococcus agalactiae have been characterized to test the immunogenicity of mastitis vaccine.
- 5. Two-dimensional gel electrophoresis: The two-dimensional polyacrylamide gel electrophoreses (2D-PAGE) is an efficient and reliable method for separation of proteins on the basis of their mass and charge. 2D-PAGE is capable of resolving ~5,000 different proteins successively, depending on the size of gel. The proteins are separated by charge in the first dimension while in second dimension separated on the basis of differences between their mass. The 2-DE is successfully applied for the characterization of post- translational modifications, mutant proteins and evaluation of metabolic pathways. Neidhardt and van Bogelen introduced the highly sensitive technique of 2-DE into the bacterial physiology. Two-dimensional differential gel electrophoresis: 2D-DIGE utilizes the proteins labeled with CyDye that can be easily visualized by exciting the dye at a specific wavelength. Cell wall proteins (CWPs) of toxic dinoflagellates Alexandrium catenella labeled with Cy3 have been identified through 2D-DIGE. Stable Isotopic Labeling with Amino Acids in Cell Culture: SILAC is an MS-based approach for quantitative proteomics that depends on metabolic labeling of whole cellular proteome. The proteomes of different cells grown in cell culture are labeled with “light” or “heavy” form of amino acids and differentiated through MS. The SILAC has been developed as an expedient technique to study the regulation of gene expression, cell signaling, posttranslational modifications. Additionally, SILAC is a vital technique for secreted pathways and secreted proteins in cell culture. SILAC was used for quantitative proteome analysis of B. subtilis in two physiological states such as growth during phosphate and succinate starvation. More than 1,500 proteins were identified and quantified in the two tested states. SILAC was used by for quantitative proteome analysis of A. thaliana. Expression of glutathione S-transferase was analyzed in response to abiotic stress due to salicylic acid and consequent proteins were quantified. The intracellular stability of almost 600 proteins from human adenocarcinoma cells have been analyzed through “dynamic SILAC” and the overall protein turnover rate was determined. Isobaric tag for relative and absolute quantitation:
- 6. iTRAQ is multiplex protein labeling technique for protein quantification based on tandem mass spectrometry. This technique relies on labeling the protein with isobaric tags (8-plex and 4-plex) for relative and absolute quantitation. The technique comprises labeling of the N-terminus and side chain amine groups of proteins, fractionated through liquid chromatography and finally analyzed through MS. It is essential to find the gene regulation to understand the disease mechanism, therefore protein quantitation using iTRAQ is an appropriate method that helps to identify and quantify the protein simultaneously. X-ray crystallography: X-ray crystallography is the most preferred technique for three dimensional structure determination of proteins. The highly purified crystallized samples are exposed to X-rays and the subsequent diffraction patterns are processed to produce information about the size of the repeating unit that forms the crystal and crystal packing symmetry. X-ray crystallography has an extensive range of applications to study the virus system, protein–nucleic acid complexes and immune complexes. Further, the three-dimensional protein structure provides detailed information about the elucidation of enzyme mechanism, drug designing, site-directed mutagenesis and protein– ligand interaction. Mass spectrometry: MS is used to measure the mass to charge ratio (m/z), therefore helpful to determine the molecular weight of proteins. The overall process comprises three steps. The molecules must be transformed to gas-phase ions in the first step, which poses a challenge for biomolecules in a liquid or solid phase. The second step involves the separation of ions on the basis of m/z values in the presence of electric or magnetic fields in a compartment known as mass analyzer. Finally, the separated ions and the amount of each species with a particular m/z value are measured. Commonly used ionization method comprises matrix-assisted laser desorption ionization (MALDI), surface enhanced laser desorption/Ionization (SELDI) and electrospray ionization (ESI).