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Proteomics Post-translational Modification Post-translational Modification Many proteins undergo chemical modifications at certain amino acid residues following translation. These modifications are essential for normal functioning of the protein and are carried out by one or more enzyme catalyzed reactions. Learning Objective In this Learning Object, the learner will be able to, 1.Recall what Post translation modifications are. 2.Describe Gel-based detection techniques for Post translation modifications. 3.Define MS-based detection techniques for Post translation modifications, 4.Describe Microarray-based detection techniques for Post translation modifications. Proteomics Post-translational Modification Overview of PTMs Once the protein has been synthesized by the ribosome from its corresponding mRNA in the cytosol, many proteins get directed towards the endoplasmic reticulum for further modification. Certain N and C terminal sequences are often cleaved in the ER after which they are modified by various enzymes at specific amino acid residues. These modified proteins then undergo proper folding to give the functional protein. Proteomics Post-translational Modification Overview of PTMs There are several types of post translational modifications that can take place at different amino acid residues. The most commonly observed PTMs include phosphorylation, glycosylation, methylation as well as hydroxylation and acylation. Many of these modifications, particularly phosphorylation, serve as regulatory mechanisms for protein action. Proteomics Post-translational Modification Overview of PTMs The final structure of functional proteins most often does not correlate directly with the corresponding gene sequence. This is due to the PTMs that occur at various amino acid residues in the protein, which cause changes in interactions between the amino acid side chains thereby modifying the protein structure. This further increases the complexity of the proteome as compared to the genome. Proteomics Post-translational Modification Overview of PTMs Phosphorylation of amino acid residues is carried out by a class of enzymes known as kinases that most commonly modify side chains of amino acids containing a hydroxyl group. Phosphorylation requires the presence of a phosphate donor molecule such as ATP, GTP or other phoshorylated substrates. Serine is the most commonly phosphorylated residue followed by threonine and tyrosine. Removal of phosphate groups is carried out by the phosphatase enzyme and thus this forms one of the most important mechanisms for regulation of proteins. Proteomics Post-translational Modification Overview of PTMs Glycosylation involves the enzymatic addition of saccharide molecules to amino acid side chains. This can be of two types – N-linked glycosylation, which links sugar residues to the amide group of aspargine and Olinked glycosylation, which links the sugar moieties to the hydroxyl groups of serine or threonine. Suitable glycosyl transferase enzymes catalyze these reactions. Sugar residues that are attached most commonly include galactose, mannose, glucose, Nacetylglucosamine, Nacetylgalactosamie as well as fucose. Proteomics Post-translational Modification Gel-based detection techniques for PTMs Protein phosphorylation can be detected using a novel gel-based detection technique. Proteins separated on a 2-DE gel are first placed in a fixing solution containing methanol and acetic acid which fixes the protein bands on to the gel and minimizes any diffusion. They are then stained using the Pro-Q-diamond staining solution which selectively stains only phosphoproteins on the gel. The excess stain is then washed off with a solution of methanol and acetic acid. Proteomics Post-translational Modification Gel-based detection techniques for PTMs The stained gel is then scanned at its excitation wavelength using a gel scanner. The gel image obtained shows the protein bands corresponding to only the phosphoproteins present. This image is saved and the gel is then removed from the scanner for treatment with the second stain, a procedure known as dual staining. Proteomics Post-translational Modification Gel-based detection techniques for PTMs The scanned gel is then removed from the scanner and placed in the SYPRORuby Red fluorescent dye solution. This dye stains all the protein spots present on the gel thereby providing a total protein image with sensitivity down to nanogram level. Excess dye is then washed off using a solution of methanol and acetic acid. Proteomics Post-translational Modification Gel-based detection techniques for PTMs The gel stained with SYPRO-Ruby Red is then scanned in the gel scanner at its excitation maxima. The image produced will have more number of spots since all proteins present on the gel are detected. This dual staining procedure provides a useful comparative profile of the phosphoproteins and the total proteins on the gel, thereby enabling detection of the phosphorylated proteins. Proteomics Post-translational Modification Gel-based detection techniques for PTMs Protein mixture containing phosphorylated as well as other unmodified proteins can be separated by a suitable electrophoresis technique. SDS-PAGE and two dimensional gel electrophoresis are most commonly used for protein separation. These separated proteins on the gel are used for further analysis. Proteomics Post-translational Modification Gel-based detection techniques for PTMs The separated protein bands are then blotted onto a nitrocellulose membrane. These membranes are then probed either by means of specific anti-phospho-amino acid antibodies or more recently, by motif antibodies that specifically bind to proteins having phosphorylation at a particular amino acid residue. This binding interaction can then be detected by means of suitably labeled secondary antibodies or by autoradiography using a radioactive probe. Thus, the use of immunoblotting technique has been shown to be extremely effective for detection of PTMs. Proteomics Post-translational Modification MS-based detection techniques for PTMs Post translational modifications can be detected by means of mass spectrometry due to the unique fragmentation patterns of phosphorylated seine and threonine residues.. The modified protein of interest is digested into smaller peptide fragments using a suitable enzyme like trypsin. This digest is then mixed with a suitable organic matrix such as -cyano-4hydroxycinnamic acid, sinapinic acid etc. and then spotted on to a MALDI plate. Proteomics Post-translational Modification MS-based detection techniques for PTMs The target plate containing the spotted matrix and analyte is placed in a vacuum chamber with high voltage and short laser pulses are applied. The laser energy gets absorbed by the matrix and is transferred to the analyte molecules which undergo rapid sublimation resulting in gas phase ions. These ions are accelerated and travel through the flight tube at different rates. The lighter ions move rapidly and reach the detector first while the heavier ions migrate slowly. The ions are resolved and detected on the basis of their m/z ratios and a mass spectrum is generated. Proteomics Post-translational Modification MS-based detection techniques for PTMs Identification of PTMs by MS largely lies in the interpretation of results. Comparison of the list of observed peptide masses from the spectrum generated with the expected peptide masses enables identification of those peptide fragments that contain any PTM due to the added mass of a modifying group. In this hypothetical example, two peptide fragments are found to have different m/z values, differing by 80 daltons and 160 daltons. It is known that the added mass of a phosphate group causes an increase in m/z of 80 daltons. Therefore, this principle of mass difference enables detection of modified fragments. Proteomics Post-translational Modification MS-based detection techniques for PTMs Liquid chromatography coupled with mass spectrometry serves as a useful technique for enrichment and identification of proteins having a particular type of PTM from a complex mixture. The complex protein sample is loaded onto a miniaturized affinity column which will interact specifically with proteins having the PTM of interest. Here, we depict the use of immobilized metal affinity chromatography columns containing ions such as Ga3+, Zn2+, Fe3+ or TiO2 which have been found to specifically chelate the phosphorylated proteins. Unwanted proteins are removed by washing the column with a suitable buffer solution after which the phosphorylated protein of interest is eluted out by modifying the buffer solution. Proteomics Post-translational Modification MS-based detection techniques for PTMs The protein purified by liquid chromatography is then subjected to typtic digestion followed by analysis using tandem mass spectrometry. Here we demonstrate the use of MALDITOF-TOF-MS for resolution of the generated ion fragments. Separation is based on the flight time of the ions and greater resolution is achieved due to the presence of two mass analyzers. The peptide ion spectrum generated is analyzed by comparing it with the expected spectrum, thereby allowing determination of modified peptides having different m/z values. Proteomics Post-translational Modification Microarray-based detection techniques for PTMs PTMs can also be detected by means of protein microarrays using a kinase assay. Potential substrates for protein phosphorylation are immobilized on a suitably coated array surface. To this, kinase enzyme and gamma P-32 labeled ATP are then added and the array is incubated at 30oC. The phosphorylation reaction occurs at those sites containing proteins that can be modified. Proteomics Post-translational Modification Microarray-based detection techniques for PTMs After sufficient incubation, excess unbound ATP and enzyme are washed off the array surface. Detection is carried out by means of autoradiography wherein a photographic film is placed in contact with the array surface. The radioactive emissions from the phosphate label present at the phosphorylated protein sites strike the film. Upon development, the positions at which phosphorylation has occurred can be clearly determined. Thus proteome chip technology offers a useful platform for detection of phosphporylated proteins. Proteomics Post-translational Modification Microarray-based detection techniques for PTMs Antibodies specific to phosphorylated serine, threonine or tyrosine residues as well as motif antibodies can be immobilized on to a suitably coated microarray surface and used for detection of PTM. The complex protein mixture containing modified and unmodified proteins is labeled with a suitable fluorescent tag molecule and added to the array surface. Specific binding interactions occur between the phosphorylated proteins and their corresponding antibodies. Proteomics Post-translational Modification Microarray-based detection techniques for PTMs The array is then washed to remove any excess unbound proteins from the surface. This is followed by scanning of the array using a microarray scanner at a suitable wavelength to detect the fluorescent tag of the bound proteins. This method offers sensitive and simultaneous detection of large number of post translationally modified proteins. Proteomics Post-translational Modification Overview of PTMs 1. Post-translational modification (PTM): The chemical modifications that take place at certain amino acid residues after the protein is synthesized by translation are known as posttranslational modifications. These are essential for normal functioning of the protein. Some of the most commonly observed PTMs include: c) Acylation: The process by which an acyl group is linked to the side chain of amino acids like aspargine, glutamine or lysine. a) Phosphorylation: The process by which a phosphate group is attached to certain amino acid side chains in the protein, most commonly serine, threonine and tyrosine. e) Hydroxylation: This PTM is most often found on proline and lysine residues which make up the collagen tissue. It enables crosslinking and therefore strengthening of the muscle fibres. b) Glycosylation: The attachment of sugar moieties to nitrogen or oxygen atoms present in the side chains of amino acids like aspargine, serine or threonine. d) Alkylation: Addition of alkyl groups, most commonly a methyl group to amino acids such as lysine or arginine. Other longer chain alkyl groups may also be attached in some cases. Proteomics Post-translational Modification Overview of PTMs 2. Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids. 3. Cytosol: A cellular compartment that serves as the site for protein synthesis. 4. Signal sequence: A sequence that helps in directing the newly synthesized polypeptide chain to its appropriate intracellular organelle. This sequence is most often cleaved following protein folding and PTM. 5. Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for post- translational modification of synthesized polypeptide chains. the newly 6. Cleaved protein: The protein product obtained after removal of certain amino acid sequences such as N- or C-terminal sequences, signal sequence etc. Proteomics Post-translational Modification Gel-based detection techniques for PTMs 1. Pro-Q-diamond: This fluorescent dye is capable of detecting modified proteins that have been phosphorylated at their serine, threonine or tyrosine residues. They are suitable for use with electrophoretic techniques or with protein microarrays and offer sensitivity down to few ng levels, depending upon the format in which they are used. This dye can also be combined with other staining procedures thereby allowing more than one detection protocol on a single gel. b) Gel scanning: The visualization of the stained protein bands on an electrophoresis gel by exciting it at a suitable maximum wavelength such that the dye absorbs the light and emits its own characteristic light at another emission wavelength. a) Gel staining: The process by which the protein bands on an electrophoresis gel are stained by suitable dyes for visualization. a) Electrophoresis: Electrophoresis is a gelbased analytical technique that is used for separation and visualization of biomolecules like DNA, RNA and proteins based on their fragment 2. Immunoblotting: This process, also known as Western blotting, is a commonly used analytical technique for detection of specific proteins in a given mixture by means of specific antibodies to the given target protein. Proteomics Post-translational Modification Gel-based detection techniques for PTMs lengths or charge-to-mass ratios using an electric field. The protein mixture is first separated by means of a suitable electrophoresis technique such as SDS-PAGE or Two-dimensional Electrophoresis. b) Blotting: The process by which the proteins separated on the electrophoresis gel are transferred on to another surface such as nitrocellulose by placing them in contact with each other. c) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which the protein bands separated by electrophoresis are transferred for further probing and analysis. d) Specific probe antibodies: Antibodies that are specific to a particular protein modification can be used as probes to detect those proteins containing that particular PTM. Protein phosphorylation is commonly detected using anti-phosphoserine, phosphothreonine or phosphotyrosine antibodies. Recently, specific motif antibodies have also been developed which detect a particular sequence of motif of the protein that contains a PTM. e) Labeled secondary Abs: Antibodies labeled with a suitable fluorescent dye molecule are used to detect the interaction between the modified protein and its antibody by binding to another domain of the probe antibody. Proteomics Post-translational Modification MS-based detection techniques for PTMs 1. MALDI-TOF-MS: A mass spectrometry instrument that produces charged molecular species in vacuum, separates them by means of electric and magnetic fields and measures the mass-to-charge ratios and relative abundances of the ions thus produced. It has the following components: a) Ion source: The ion or ionization source is responsible for converting analyte molecules into gas phase ions in vacuum. The technology that enables this is termed soft ionization for its ability to ionize non-volatile biomolecules while ensuring minimal fragmentation and thus, easier interpretation. In MALDI-TOF-MS, the ion source used is MALDI, in which the target analyte is embedded in dried matrix- sample and exposed to short, intense pulses from a UV laser. b) Flight tube: Connecting tube between the ion source and detector within which the ions of different size and charge migrate to reach the detector. The Time-of-Flight mass analyzer correlates the flight time of the ion from the source to the detector with the m/z of the ion. c) Detector: The ion detector determines the mass of ions that are resolved by the mass analyzer and generates data which is then analyzed. The electron multiplier is the most commonly used detection technique. Proteomics Post-translational Modification MS-based detection techniques for PTMs 2. LC-MS/MS approach: LC-MS/MS a common analytical tool that combines physical separation by liquid chomatography with mass analysis and resolution by mass spectrometry. It is capable of separating and identifying complex mixtures for proteomics studies. a) Liquid chromatography: This is a chromatographic separation technique that separates molecules based on their differential adsorption and desorption between the stationary matrix phase in the column and the mobile phase. b) Affinity columns: Columns that make use of specific affinity interactions between the analyte of interest and the bound stationary phase matrix thereby successfully separating this component from a complex mixture. Immobilized Metal ion Affinity Chromatography (IMAC) is one such affinity technique that relies on the formation of specific coordinate-covalent bonds between certain amino acid residues of the protein (like histidine) and the immobilized metal ions. Phosphorylated proteins have been found to bind specifically to ions such as iron, gallium and zinc, thus facilitating their separation by IMAC. Recently, titanium dioxide (TiO2) columns have proved to be extremely useful for specific separation of phosphorylated proteins. Proteomics Post-translational Modification MS-based detection techniques for PTMs c) Tandem MS: This is a mass spectrometry technique that makes use of a combination of ion source and two mass analyzers, separated by a collision cell, in order to provide improved resolution of the fragment ions. The mass analyzers may either be the same or different. The first mass analyzer usually operates in a scanning mode in order to select only a particular ion which is further fragmented and resolved in the second analyzer. This can be used for protein sequencing studies. Proteomics Post-translational Modification Microarray-based detection techniques for PTMs 1. Protein microarrays: These are miniaturized arrays normally made of glass, onto which small quantities of many proteins can be simultaneously immobilized and analyzed. For detection of phosphorylation sites, potential protein substrates are immobilized on to the array. a) Kinase enzyme: An enzyme that is responsible for phosphorylation of specific amino acid residues in the protein with the help of ATP as a phosphate donor. b) Phosphorylated proteins: Proteins that have been phosphorylated at specific amino acid residues. c) Autoradiography: Radioactivity is the process by which certain elements spontaneously emit energy in the form of particles or waves due to disintegration of the unstable atomic nuclei into a more stable form. These radiations that are given out can be detected by means of autoradiography, wherein the radiations are allowed to strike a photographic film which on exposure shows the presence radioactive emissions. 2. Antibody microarrays: An array onto which different antibodies are spotted, which have specific binding domains for detection of the protein of interest from a complex mixture. For detection of PTMs, antibodies against specific protein motifs containing the PTM or against a Proteomics Post-translational Modification Microarray-based detection techniques for PTMs specific residue containing a phosphorylated site may be used. a) Labeled protein mixture: The protein mixture containing the protein of interest is labeled uniformly with a suitable fluorescent dye which can be detected by scanning at the appropriate wavelength. Cyanine dyes are commonly used for such labeling purposes. b) Array scanning: Once the binding interactions have taken place on the array surface and excess unbound material has been washed away, the array is scanned using a microarray scanner. This scans the array at a suitable wavelength depending upon the fluorescent dye used for labeling purposes to generate an image depicting the array positions at which binding has occurred. Books