Experiment 8: Analysis of Post-Translational Modifications
Objective: To study different post translational modifications in gliomas using tumor tissues
Post translational modifications are ubiquitous and dynamic additions in the protein structures which alters their structural and functional properties. These are the covalent processing events that alter the properties of a protein by addition of different chemical groups to one or more amino acids or proteolytic cleavage of proteins. These chemical additions, apart from being “decoration” entities, also define the physical and biochemical state of a protein in a specific location and time in the cell. For instance, the action of the enzyme kinases in the cell is to add phosphate groups to the proteins (enzymes) which at times either activates or deactivates its enzymatic activity. Such modifications in the protein structures which occur after translation are known as post translational modifications and are important to maintain the regulatory cycle for different cellular and molecular activities. The increase in complexity of the proteome is attributed to these chemical alterations and they play a key role in functional proteomics. The progress in proteomics is attaining success in unravelling these minute alterations and their detailed structural elucidation for a better functional insight in the involved pathways.
It is challenging to study these intricacies at sub-cellular level as every PTM happening in the cell is a controlled by complex regulatory pathways. Each such pathway is initiated by different intracellular/extracellular proteins. These proteins are further controlled, either by other regulatory pathways or they are the external products or allergens. Thus targeting a single molecular modification will lead to examination of an array of other proteins involved in that pathway. Study of PTMs in gliomas is expected to reveal different phosphorylations and glycosylations that happen majorly to regulate the events like cell cycle, cell division, signalling pathways, and other biological processes. A lot of phosphorylated proteins are anyways observed on the 2-DE and 2D-DIGE gels as beaded appearances. A few test gels have these new appearances on a couple of proteins when compared to the control gels which can be interpreted by the modification of these proteins in the diseased condition while a couple of proteins show these appearances on both test and control gels signifying the permanent phosphorylation of these proteins in the biological processes. For the proteomic analysis, a greater conconcentration of the proteins is needed to sufficiently target the PTMs of interest for specific proteins that can be well identified, studied and validated.
Post translational modifications of proteins being the structural alterations can be of diverse nature. Different chemical molecules may interact with proteins and modify them at different locations. Studying the PTMs in a diseased condition can relate to the molecular alterations of proteins which can be a root cause of some of the major disturbances in the protein pool which affect the normal functioning of the body machinery. The most common PTMs are:
Reversible protein phosphorylation is the most common process that regulates many cellular and molecular events by modifying either or many of the Serine, Threonine or Tyrosine residues. It plays a critical role in different processes like signal transduction pathways, apoptosis, cell cycle and growth. Kinases are one class of enzymes that are involved in transferring a phosphate group to a protein molecule and Phosphatases reverse the reaction by removing the phosphate group. They work in parallel to functionally regulate single or a goup of proteins targeting specific functions in the body.
Nitrosylation refers to the addition of the nitric oxide moieties to the thiol groups which are formed from cysteine residues. This leads to the formation of S-nitrosothiol or SNOs which are used to stabilize the protein structures and regulate the gene expression in various biological pathways. These nitrosylation take place at selected cysteine residues in the proteins making it more selective. These events are also reversible as the de-nitrosylation removes these NO groups when not required by other enzymes.
Glycosylation refers to the addition of carbohydrate moieties to the protein molecules by the formation of glycosidic linkages that is utilized in various conformational changes, protein folding and stability. It is one of the major PTM which regulates the activity of various proteins in different body systems. The modifications can be either single monosaccharide additions to smaller nuclear proteins or incorporation of large polysaccharides to the cell surface receptors. The most common among these is the N-linked glycosylation (Asparagine-linked) which occurs in the eukaryotes for cell-cell and cell-extracellular matrix interactions. O-linked glycosylation (Serine/Threonine linked) are also the most prevalent forms that are engaged in the structural components of major surface proteins and receptors.
Acetylation refers to the addition of the acetyl groups to the N-terminal amino acids where N-terminal Amino peptidase cleaves the N-terminal methionine and further the acetyl groups are added there by the action of N-acetyl transferases. These acetylations play a huge role in different biological processes ranging from protein regulation to protein translation to control of various immunological functions.
The binding of the ubiquitin molecules to the target proteins that need to be recycled is referred to as ubiquitination. Ubiquitin is a small polypeptide made up of 76 amino acids and 8kDa molecular weight. Ubiquitination involves the formation of an amide bond between the carboxylic acid of the terminal glycine of the activated ubiquitin and the epsilon amine of the lysine of the target protein. Single or monoubiquitination is followed by addition of ubiquitin molecules to form polyubiquitinated proteins that are recognized by the 26S proteasomes that degrade these proteins and recycle the ubiquitin molecules.
Lipidation refers to the process of addition of the lipid moieties to the amino acids to anchor them to the hydrophobic surfaces like lipid membranes for targeted functions. Lipid addition increases the hydrophobicity of the membrane and enhances targeted functions. The four broad categories of lipodation are: S-myristoylation, N-terminal myristoylation, GPI anchors and S-prenylation.
Methylation refers to the addition of methyl (-CH3) groups to the Nitrogen or Oxygen moieties of the amino acids, which is referred to as N or O- methylation, respectively. Methyl group, being positively charged, imparts an extra charge to the proteins which is used to neutralize any negatively charged groups. It also increases the hydrophobicity of the proteins. O-methylation is reversible while when compared to N-Methylation. Methylation is widely observed in histones where the histones are methylated and de-methylated for their availability to DNA replication machinery.
Proteolysis refers to the cleavage of the highly stable peptide bonds with the release of huge amount of energy. These processes are involved in wide range of biological processes to facilitate specific tasks like antibody synthesis, apoptosis, immunological combat to antigens and a range of other processes.
The advancements in proteomics is been utilized efficiently to study different PTMs using the 2-DE, 2D-DIGE, SDS-PAGE, Western Blotting and Mass Spectrometric approaches. Here we will concentrate on the two most common PTMs; Phosphorylation and Glycosylation using the 2DE and 1D approach separately followed by Mass spectrometry. To gain the complete understanding of this experiment, we suggest the study of the given sections in the following order:
I. Protein extraction and Quantification
II. 1D-SDS PAGE
IV. In gel digestion & Mass Spectrometry
I. Protein Extraction and Quantification
Tumor tissues are processed for protein extraction by TRIzol protein extraction method. This method separates the DNA, RNA and proteins based on the phase separation phenomena. Refer to Module V for information. The proteins are quantified and required concentration is taken for further experimentation.
II. 1D SDS_PAGE
The glioma and normal protein samples are separated on small 7cm gels under a constant electric voltage. The samples mixed with Laemmlli buffer are separated on 12.5% poly-acrylamide gels under a constant voltage of 100V. The two gels are stained with the specific dyes corresponding to different PTMs when compared to each other (as explained later).
Required concentration of glioma and control samples are rehydrated separately on IPG strips of required length and pH. The proteins are reduced by DTT and rehydrated on the IPG strips of selected pH range and length by passive or active absorption. The strips absorb and retain the proteins for further separation.
IEF or Iso-electric focusing includes the separation of the proteins based on their pI values on IPG strips or Immobilized pH gradient strips. These strips are subjected to increasing voltage to remove the salts in the initial run and finally separate the proteins where the proteins migrate on the strips based on their charge under the effect of high voltage and stop and concentrate at the pH values where their net charge is zero (Isoelectric pH).
Equilibration refers to reduction and alkylation of the cysteine residues which leads to the primary peptides and prevent them from folding back to form disulphide. Equilibration with Buffer I (containing DTT) breaks all the disulphide linkages in between the peptides to yield primary peptide chains while equilibration with Buffer II (containing IAA) leads to alkylation of the cysteine residues to prevent the re-formation of the disulphide linkages. This leads to linear peptides which are used further for SDS-PAGE.
The second dimension or SDS-PAGE separates the previously separated proteins on the basis of their molecular weights with the High Molecular weight proteins migrating slower and Low molecular weight proteins migrating faster. The proteins are separated under denaturing conditions usually on a 12.5% gel. The gels are stained with specific dyes which correspond to each PTM. The excessive stain is removed and the gels are scanned and analyzed.
Figure : Schematic for PTM specific staining and analysis
IV. In-gel digestion & Mass Spectrometry
The spots and bands are anaylsed from 1D and 2-DE and interpretations are made. The selected spots are confirmed for their identity further. For that, the selected spots on 2-DE are cut and the protein is digested into smaller peptides by trypsinization and extracted for characterization. The proteins are subjected to MALDI-TOF/TOF analysis and the identified sequences are confirmed for their identity using MASCOT.