. .
.
Experiment-7 : Studying protein interactions
.
.

 

 

 

Experiment-7: Studying protein interactions
 
Objective: To identify novel protein-protein interactions in a cancer cell line.
Several methods can be used to investigate protein-protein interactions. Each of these methods has its own strengths and weaknesses, especially with regard to its sensitivity and specificity. The choice of methods would depend on the aim of the experiment. Two of the commonly used methods for studying protein interactions are co-immunoprecipitation and Pull-down assay. Since both techniques have wide applicability, each of them is described in this experiment. To gain complete knowledge of the experiments defined in this website, it is important to understand each of the following sections. Hence, we recommend that the content of each section be read in the given order.
SECTION I: Studying protein interactions using co-immunoprecipitation
  • Sample preparation: Total protein content of the cell is extracted from breast cancer cell line.
  • Co-immunoprecipitation
  • 2DE: Protein precipitate is then quantified before rehydration of the IPG strips to carry out 2DE.
  • Staining and scanning of the blot: This provides the image needed to carry out analysis.
  • Software analysis: This helps to identify the protein spots on the gel.
  • In-gel digestion of the excised spots
  • MALDI-TOF/TOF and MASCOT analysis: This provides the identity of the protein.
SECTION II: Studying protein interactions using pull-down assay
  • Sample preparation: Total protein content of the cell is extracted from breast cancer cell line.
  • Pull-down assay:
  • 2DE: Protein precipitate is then quantified before rehydration of the IPG strips to carry out 2DE.
  • Staining and scanning of the blot: This provides the image needed to carry out analysis.
  • Software analysis: This helps to identify the protein spots on the gel.
  • In-gel digestion of the excised spots
  • MALDI-TOF/TOF and MASCOT analysis: This provides the identity of the protein.

After the details of the technique are understood, the reader is encouraged to go through the stimulations, protocols and manuals to get better insight of the process.

 

 

Theory: 
Most proteins usually function as a part of a larger protein complex. Discovering the protein-protein interactions that make up such complexes is important for studying protein function and understanding the resulting cellular processes. If a previously unknown protein is observed to interact with other proteins or a complex of known function, then its function can be predicted. By studying the ways in which proteins react with each other, researchers aim to achieve two goals: learn more about the ways in which cells function, and develop new drugs to treat disease.  Moreover, genome-wide elucidation of protein-protein interactions yields a network structure that may result in higher level information about interactions within and among protein complexes.
Proteins bind to each other at specific binding domains with the help of a combination of Van der Waals forces, hydrophobic bonding and salt bridges. Such binding domains could be small binding clefts or large surfaces, comprising of only a few amino acid sequences or a number of large peptides. The size of the binding domain influences the strength of the protein-protein binding. 
Protein interactions can be broadly divided into two types, viz., stable and transient. Stable interactions are usually seen between proteins that make up a multi-subunit complex. Proteins in such a complex could be identical or different. Examples of stable protein-protein interactions are leucine zipper, hemoglobin and core RNA polymerase. These interactions can be either strong or weak. Such interactions are easy to isolate by physical methods like co-immunoprecipitation (co-IP), pull-down assay or far-Western methods since the protein complex does not disassemble over time. 

On the other hand , transient interactions are temporary in nature and commonly occur between binding domains of signalling proteins like the Src homology (SH) domains , SH2 and SH3.They require a set of conditions that promote that promote the interaction, such as phosphorylation, conformational changes or localization to discrete areas of the cell .While in contact with their binding partners , transiently interacting proteins are expected to be involved in a whole range of cellular processes including protein modification , transport , folding , signalling , foding , signalling , cell cycling , etc. Such reactions can be strong or weak , fast or slow. They can be identified by covalently crosslinking the proteins to freeze the interaction, followed by Co-IP or pull -down assay. Alternatively , crosslinking , along with lable transfer and far-western blot analysis can be performed. 

SECTION I: Studying protein interactions using Co-immunoprecipitation
1.     Sample preparation and quantification
Breast cancer cell line is grown to mid-log phase under conditions that are explained in detail in Experiment 1. The cells are harvested under sterile conditions and lysed using a lysis buffer and mild sonication. Proteins are then isolated from the cell lysate by the Trizol method. The exact concentration of proteins in the solution is then determined using the Bradford method.
2.     Co-immunoprecipitation (co-IP)
Co-immunoprecipitation is considered to be the gold standard assay for protein–protein interactions, especially when it is performed with endogenous (not overexpressed or tagged) proteins. It is conducted in essentially the same manner as an immunoprecipitation (IP) of a single protein, in which the protein of interest is isolated with a specific antibody. The only difference is that the antigen (or “bait”) precipitated by the antibody “co-precipitates” a binding partner/protein complex (also called as “prey”) from a lysate. In brief, the interacting protein is bound to the antigen, which is bound by the antibody that is immobilized to the support. Immunoprecipitated proteins and their binding partners can then be detected by two dimensional gel electrophoresis (2DE). The assumption that is usually made when associated proteins are co-precipitated is that these proteins are related to the function of the target antigen at the cellular level. This is only an assumption, however, that is subject to further verification. However, it must be borne in mind that immunoprecipitation experiments reveal direct and indirect interactions. Hence, positive results may indicate that two proteins interact directly or may interact via one or more bridging molecules like bridging proteins, nucleic acids (DNA or RNA), or other molecules.
 

       
3.    2-D gel electrophoresis:
Two-dimensional (2-D) gel electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures. It separates proteins according to two independent properties, in two separate steps. The first step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI), while the second step, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights. Proteins and/or polypeptide chains which form a complex can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each constituent protein or polypeptide chain in the complex can be obtained. This method is explained in detail in Experiment 1.
4.    Staining and scanning of the gel:
After the electrophoretic run is completed, the separation patterns of the protein samples are visualized by staining the gels. This is done by exposing the gels to specific dyes which bind to proteins embed in the gels and help visualization of maximum number of protein spots. Commonly used dyes are Comassie brilliant blue, silver stain and Sypro Ruby stain among others. Selection of the appropriate dye depends on the overall objective of the experiment. 
Gels are then scanned using a gel documentation instrument which usually consists of a chamber and a detector. The gel is place on the imaging platform, taking care that it does not break during the transfer. An image of the gel is captured and stored with an appropriate lable. Such images of the stained gels can then be used for comparison of the global expression profiling of proteins across different gels with the help of commercially available software.
5.    Software analysis: 
Scanned images of 2-D electrophoresis gels show several spots, each spot representing a single protein or a group of protein isoforms, having a particular pI and molecular weight. With the help of commercially available software, each spot is defined by an outline which is automatically or manually drawn around it. The software then digitizes the image file into pixels. The sum of the intensitites of all the pixels present within the defined region of a spot is recorded and co-related with the quanitity of proteins present in each spot. Such an analysis gives a comprehensive output in statistical terms, which are easy to interpret. One such software, ImageMaster 2D Platinum, is explained in detail in Experiment 1.
6.     In-gel digestion
In-gel proteolytic digestion of separated proteins is performed to cleave the protein of interest present within the polyacrylamide matrix and generate a mixture of its constituent peptides. These peptides are then identified by mass spectrometry. In-gel digestion is a multi-step procedure, which include spot selection, spot excision, stain removal, reduction, alkylation, proteolytic cleavage and finally extraction of the peptides.  The spot of interest is first excised from the gel in an automated or manual way. The gel plugs are washed with a mixture of bicarbonate buffer and acetonitrile for removal of the staining agent. Acetonitrile  reduces the hydrophobic interaction between protein and the stain, while the ionic solution decreases the ionic interaction between negatively charged dye and positively charged protein. The next step includes the reduction and alkylation of the protein residues to denature the protein into its primary structure. Treatment of the protein residues with DTT breaks the disulfide bonds completely. However, reformation of disulfide bonds may occur. To prevent this, IAA an alkylating agent, is used. It adds acetoamide group to the sulfhydral group and prevents the disulfide bond formation. The proteins are then digested to generate peptides. Several proteolytic enzymes like chymotrypsin, trypsin, pepsin are used for this purpose. CNBr cleaves the peptide bond at Methionine residue, while trypsin breaks the peptide bond at carboxyl terminal of basic amino acids such as arginine and lysine.
 
Figure 2. Tryptic digestion of protein residues. Trypsin recognizes the lysine and arginine resides and cleaves to generate smaller peptide fragments.
After overnight incubation, peptides generated through proteolytic digestion are extracted using extraction buffer containing 0.1% FA/TFA in 50% ACN solution. Efficient extraction process is essential to ensure the release of peptides from gel-matrix to solution, which is further subjected to mass spectrometric analysis.     
7.     MALDI-TOF/TOF analysis:
 Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a commonly used mass spectrometer configuration. MALDI is an efficient process for generating gas-phase ion of peptides and proteins for mass spectrometric detection by exposing the target analyte to short, intense pulses of laser. TOF is a mass analyzer in which the flight time of the ion from the source to the detector is correlated to the m/z of the ion. MALDI-TOF is widely used in proteomic research as a high-throughput technique to identify proteins and their post translational modifications. It is also applicable for detection of intact molecular weight of any analytes.
 
Figure 3. Schematic representation of the three components of mass spectrometer. Ionization source to generate analyte ions, which start moving in the mass analyzer to get separated from each other, depending upon the mass/charge ratio, and the ion detector in which the analyte ions are detected.
 
Just like any other mass spectrometer, MALDI-TOF has three basic components, viz., the ionization source, the mass analyzer and the detector. MALDI is one of the most widely used soft desorption ionization source for mass spectrometric analyses. A chemical compound referred as matrix is used for ionization of the sample.  Matrix molecules have a specialized property of getting excited to a higher energy state when encountered with a UV-laser beam. Thus while coming back to the ground state they concomitantly give out energy which in turn is received by the sample molecules, that leads to the formation the ions of interest. Generated ions may receive a single proton and form [M+H]+.  Ionized molecules enter the mass analyzer and form the mass spectrum. The whole process of ionization and separation of ions takes place in high energy vacuum. There are various kinds of matrices commercially available. Usually these matrices are low molecular weight aromatic compounds and interfere less with sample molecules. Selection of the matrix depends upon the sample to be analyzed.
Once the ions are generated, they pass through the mass analyzer, which in this case is the Time of Flight. It acts in vaccum so that the highly reactive ions do not cross react or collide with other ions moving simultaneously in the space. This ensures the ions are separated to give accurate results on basis of the flight times taken by the ions to travel between two points. All the ions are accelerated at a same time and it is taken into consideration that all the released ions have same kinetic energy. Depending upon the m/z ratio, lighter ions reach the target first and heavier reach late.
Since the process of ionization and separation in mass analyzers happens within a few nano seconds, it must end with an efficient detection of the separated molecules/ions.  The third component of a mass spectrometer, called detector performs this task of detecting ions separated on the basis of charge, mass or velocity. Its main function is to get the information from mass analyzer and convert those into electric signals which are then multiplied by the photo-multiplier tubes. Ultimately, these signals are read by the data system to bring out the mass spectrum. The spectrum is then matched with those available in the database using a search engine like MASCOT, SEQUEST or ProFound for accurate identification of the protein.
 
 
SECTION II: Studying protein interactions using Pull-down assay
1.     Sample preparation and quantification
Breast cancer cell line is grown to mid-log phase under conditions that are explained in detail in Experiment 1. The cells are harvested under sterile conditions and lysed using a lysis buffer and mild sonication. Proteins are then isolated from the cell lysate by the Trizol method. The exact concentration of proteins in the solution is then determined using the Bradford method.
2.     Pull-Down Assays
Pull-down assays are similar in methodology to co-immunoprecipitation because of the use of beaded support to purify interacting proteins. The difference between these two approaches is that while co-IP uses antibodies to capture protein complexes, pull-down assays use a purified and tagged "bait" protein to precipitate any proteins in a lysate that bind to it. In this assay, a tagged bait protein is captured on an immobilized affinity ligand specific for the tag. Cell lysate is then incubated or percolated through the resulting column. The prey proteins bind to the column and are eluted using an eluting buffer.  To obtain biologically significant results using pul-down assays, it is important to carefully design control experiments. A negative control consisting of a non-treated affinity support (minus bait protein sample, plus prey protein sample) helps to identify and eliminate false-positives caused by nonspecific binding of proteins to the affinity support. The immobilized bait control (plus bait protein sample, minus prey protein sample) helps identify and eliminate false-positives caused by nonspecific binding of proteins to the tag of the bait protein. The immobilized bait control also serves as a positive control to verify that the affinity support is functional for capturing the tagged bait protein.  Pull-down assays are ideal for studying strong or stable interactions or those for which no antibody is available for co-immunoprecipitation.
 
 

 

 

 

 

Cite this Simulator:

.....
..... .....
Copyright @ 2017 Under the NME ICT initiative of MHRD (Licensing Terms)
 Powered by AmritaVirtual Lab Collaborative Platform [ Ver 00.10. ]