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Experiment-1 : Global Proteomic Analysis
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Objective: To study the global proteomic profile of a Breast cancer cell line.
 
 
Theory: 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.
 
  • Culturing of breast cancer cell line : Conditions and parameters for optimal growth of the cell line are described.

  • Sample preparation : Total protein content of the cell is extracted and the concentration of protein is evaluated.

  • Rehydration of IPG Strips : Required amount of the protein sample is measured and incubate the strip with the sample overnight.

  • IEF : This is the first dimension wherein sample protein are separated on the IPG strips on the basis of their isoelectric point.

  • Equilibration of the strips : The IPG strips are saturated with SDS buffer system required for the second - dimension separation.

  • SDS - PAGE : This is the second dimension wherein the proteins are further separated based on their molecular weight.

  • Staining and scanning of the gel : This provides the image needed to carry out analysis.

  • Software Analysis : This helps to identify the global expression pattern of protein spots on the gel.

 

After the details of the technique are understood, the reader is encouraged to go through the simulations, protocols and manuals to get better insight of the process.
        
 
Global proteome analysis is a direct representation of the total number of proteins in a system, in terms of presence and abundance at a specific point of time, under a defined set of conditions. The system can be tissues, cells, plasma/serum or a particular organelle, whose protein expression profile needs to be studied. The global proteomic study is very important to understand the physiology of the organism at a particular time point and provides insight into the response of the organism to the particular condition. In case of clinical studies, global proteomics can be studied to determine the pattern of protein expression in presence or absence of certain parameters. Proteins which play an important role in the growth and/or survival of a cell can be identified and their function in the cell can be ascertained. Unique proteins, which are expressed by the cell only under certain conditions like stress, can also be identified.
One of the popular methods used to conduct such a global proteomic analysis is called two-dimensional (2-D) gel electrophoresis. It is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. 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. Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein can be obtained. Moreover, the technique is also unique in its ability to detect post- and co-translational modifications, which cannot be predicted from the genome sequence.
 
 1.Culturing of Breast cancer cell line:
 
Cultured human cells are widely used as model systems to study various processes and pathways due to their biological relevance. They provide a simple model to understand basic biological process in the complex human system and also help to gain insight into the function of a gene at the protein level. Diverse cellular mechanisms like signal transduction, cell-cycle control, protein secretion, apoptosis and tissue specific differentiation to oncogenic transformation are being elucidated with the help of such cultured cells. A major chunk of biological research is targeted towards finding various markers for quick screening of different diseases. Since cell lines can be grown quickly on defined media under strictly controlled conditions, they are of fundamental importance in such studies. As these cell lines originate from humans, the concern of phylogenetic distance between model organism and humans is taken care of and correct interpretation of the exact mechanisms can be done.  In contrast to the entire human system, which constitutes a wide range of different cell types, human cell lines, no matter what their tissue origins are, comprise a phenotypically and genetically uniform population. Since they are grown under defined conditions, variations in external environment are negligible. Moreover, they are stable under proper culture conditions and do not enter an altered pathway without induction. Due to these characteristics, it is easy to get high-throughput data from these samples. Global proteome analysis plays an important role in identifying the essential set of proteins responsible for the growth and maintenance of cells in the culture and also in identifying the trend of protein expression under various conditions of growth.
Some of the highly studied human cell lines are those that are established from human tumors. Amongst the various tumors that can develop in a human system, breast cancinoma posses an intriguing question to the scientific community. It is the second leading cause of cancer death in women and shows large variations in the incidence, mortality and survival between different countries and regions, and even within specific regions. A number of complex factors underlie these variations like the age, race, ethinicity, lifestyle, environment and socioeconomic status of the affected person. Breast cancer cell lines are commonly used as cellular model for discovering markers. Therefore, a better knowledge of their proteome is a prerequisite for a more efficient use of this model. In the current experiment, we grow a breast cancer cell line in an artificial, well-defined culture media, providing the necessary nutrients, defining the parameters and maintaining sterile optimal conditions for growth. At the mid-log phase, cells are harvested under sterile conditions, protein extraction is carried out and 2-D electrophoresis run is planned to check and quantify the protein expression profile. 
 
 2.Sample preparation:
Breast cancer cell line is cultured in two 75 cm2 dishes containing freshly prepared minimum essential medium, supplemented with serum, insulin and antibiotic solution. Cells are allowed to grow at 37°C in a 5% CO2 atmosphere with 95% humidity. When the cells reach the exponential phase of growth and show around 70% confluency, cells are scraped off the flask in the presence of PBS buffer. Cell pellet is then washed with the same buffer and re-suspended in a lysis buffer containing the protease inhibitor cocktail to prevent protein degradation during cell lysis. The resulting mix is incubated on ice for 1 h with intermittent vortexing. In order to lyse the cells completely and release all the complex proteins present within each cell, the resulting solution is subjected to mild sonication. Further isolation of proteins from the whole cell extract can be achieved using TCA-acetone precipitation, acetone precipitation, phenol/methanol-ammonium acetate precipitation or isopropanol precipitation. However, in this experiment, we have described the use of Trizol reagent for this purpose since it simultaneously extracts RNA, DNA and proteins from the same sample. Trizol reagent contains phenol and guanidine isothiocyanate which helps in liquid-liquid phase extraction, where DNA, RNA and protein get separated into three distinct layers depending on their solubilization properties. The layer containing the protein content is treated with acetone for precipitation and pellet formed can be stored for further use. Details of sonication and subsequent protein extraction from bacterial cell extracts are explained in the procedure section of this experiment. Once a pure preparation of protein, devoid of any impurities, is obtained, the exact concentration of proteins in the solution is determined. The method for quantifying the proteins in the sample is chosen based  on their compatibility with the reagents used in the sample solution. The standard range is set to a higher extent, in order to accommodate the absorbance reading for unknown samples. The process is carried out in duplicates for standards and samples to get accurate result outputs. In this experiment, we use the modified Bradford method for protein quantification. The Bardford reagent contains Coomassie Brilliant Blue G-250 dye which shifts its absorbance maxima from 470 nm to 595 nm after binding with protein (Fig. 1). This property of the dye is used in the assay of protein quantification.

 

Fig. 1: Chemistry of protein quantification: The dye Coomassie Brilliant Blue G-250, present in the Bradford reagent, is red in color in the unbound state and has an absorbance maximum of 470 nm. After coming in contact with protein, it tends to establish non-covalent interactions with the protein to form a blue colored complex, having absorbance maxima of 595 nm.
 
 
3. Rehydration of the strips:
IPG strips are used to separate proteins during the first dimension of 2-D gel electrophoresis, i.e. IEF. They provide an immobilized pH gradient, formed by the copolymerizing buffering and titrant groups of acrylamido derivatives into a polyacrylamide gel. The IPG strips need to be rehydrated with sample protein solution along with the rehydration buffer before carrying out IEF. The rehydration buffer consists of urea, detergent, IPG buffer and dye. Urea helps in protein solubilization, detergent minimizes protein aggregation, IPG buffer improves separation and dye is used for tracking. The choice of the constituents and make of rehydration solution depends on the sample solubility properties. The volume of the rehydration solution, which now consists of sample solution and rehydration buffer, depends on the length of the IPG strip being used. This rehydration solution is added to a well in the reswell tray and the IPG strip is placed on it such that the gel side is in contact with the solution (Fig. 2). The well is then filled with a cover fluid which provides prefect condition for rehydration and avoids strip drying and sample precipitation. The rehydration process is carried out overnight or  for 10-20hrs.

 

Fig. 2: IPG strip rehydration: Sample is added to one of the wells of the reswelling tray. IPG strip is placed over the sample such that the gel side is in touch with the sample. Cover fluid is added over the strip to prevent gel drying and sample precipitation.
                                         
 
4. First dimension-IEF:
In isoelectric focusing, proteins are separated on the basis of their isoelectric point (pI). Proteins are amphoteric in nature, possessing a net charge depending on the pH of their surroundings and the sum of negative and positive amino acid side chains present on them. The specific pH at which the net charge on the protein is zero is called its pI or Iso-electric point. Proteins are negative charged at pH values above their pI and positively charged at pH values below their pI. In the presence of a pH gradient and under the influence of an electric field, a protein tends to move to a positon in the gradient where its attains its pI. IPG strips provide such an immobilized pH gradient. Druing IEF, positively charged proteins tend to migrate towards the cathode, losing their positive charge as they move down the IPG strip to attain their respective pI (Fig. 3). Similarly, negatively charged proteins become less negative and start moving towards the anode to attain their pI.  The process is carried out under constant voltage conditions in a stepped manner, initially applying a low voltage to avoid protein aggregation and precipitation, followed by maximum voltage at which proteins get resolved properly from each other. Even if a protein diffuses away from its pI, it gains a charge immediately and migrates back to its pI. This is called focusing effect of IEF.
 
Fig. 3: Isoelectric focusing: A representative image of a protein is shown to have a net positive charge at pH 4.0. Under the influence of an electric field, the protein moves along the pH gradient towards the cathode and stabilizes at a point where the net charge on it becomes zero.
 
 
 5.  Equilibration of the IPG strips:
The equilibration step for focused IPG strip treatment is carried out before subjecting the strip to the second dimension of 2-D gel electrophoresis. The IPG strip is made ready by treating with equilibration buffer, which contains urea, glycerol and SDS. Urea and glycerol counter the effect of electroendosmosis and improve the transfer of proteins from strip to SDS-PAGE gel. SDS denatures the proteins to form protein-SDS complexes. The equilibration step is carried out in two steps for 15min each, initially by adding dithiothreitol (DTT) and later by adding 2-Iodoacetamide (IAA) to the equilibration solution (Fig. 4). DTT keeps the proteins in a denatured state, while IAA prevents reoxidation by alkylating the thiol groups on protein. An appropriate amount of equilibration solution with proper treatment will help saturate the IPG strip with SDS which is required to carry out separation in the second dimension of 2-D gel electrophoresis.
 
Fig. 4: IPG strip equilibration: Schematic representation of the equilibration step is shown. A) DTT, added to the equilibration buffer, breaks the disulfide bonds present in the proteins, thereby denaturing the protein. B) IAA is then added to fresh equilibration buffer to prevent reoxidation by alkylating the thiol groups on the protein.
 
 
6.  Second dimension - SDS-PAGE:
The second dimension of 2-D gel electrophoresis is Sodium Dodecyl Sulphate - Polyacrylamide gel electrophoresis (SDS-PAGE). In this step, the proteins are separated under the applied electric field on the basis of their molecular mass/weight.   After the equilibration treatment, the proteins are in the denatured state as they form complexes with SDS. Each complex has a necklace-like structure, aiding large amounts of SDS to be incorporated, at a ratio of approximately 1.4g SDS/g protein. The SDS masks the charge present on the proteins, giving roughly a constant net negative charge per unit mass to all the protein-SDS complexes. This ensures that the electrophoresis carried out in the SDS-PAGE gel is based only on the molecular weight of the protein (Fig. 5).   The negative charged protein-SDS complex tends to move towards the anode in the presence of a buffer system. A routinely used buffer system is the Tris-glycine system reported by Laemmli, which separates proteins at high pH, and prevents protein aggregation, ensuring clear separation. The proteins get separated depending upon the mass, where in higher molecular weight proteins are separated on the top of the gel followed with gradual separation of low molecular weight down the gel.
Fig. 5: SDS-PAGE: Proteins separated on the IPG strips on the basis of their charge are coated with SDS to give them a uniform negative charge. In the presence of an electric field, these proteins then get separated only on the basis of their molecular weights.
 
7.     Staining and Scanning of 2-D electrophoresis gels:
After the second dimension of 2-D electrophoresis is done, 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. For example, if identity of the protein spots needs to be established by MS analysis then Comassie brilliant blue stain is preferred over silver stain despite the sensitivity of the later being higher by ten-fold. Coomassie dye interacts with the proteins embedded in the gel by non-covalent forces like electrostatic and Vander Walls interactions. Dye that is not bound to the protein diffuse out of the gel during destaining step. The proteins then appear as blue spots or bands on a clear background. Gels are then subjected to a corresponding destaining step before they are scanned using a gel documentation instrument. This instrument usually consists of a chamber and a detector which can capture an image of the stained gel. 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. A representative image is shown in Fig.6. 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.
 
 
Fig. 6: 2-D electrophoresis gel image: Image of a typical 2-D electrophoresis gel showing breast cancer cell line proteins separated on a 4-7 pH range on the X axis and molecular weight on the Y axis.
 
8.     Software analysis
Scanned images of 2-D electrophoresis gels show thousands of 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 intensities of all the pixels present within the defined region of a spot is recorded and co-related with the quantity of proteins present in each spot. Such an analysis gives a comprehensive output in statistical terms, which are easy to interpret and can be extended to a wider biological scenario. One such software is ImageMaster 2D Platinum, a high-throughput 2-D imaging software for almost parameter-free spot detection. Although it contains several tools which need to be explored in detail, the basic steps for image analysis are as follows:
  • A master folder is first created. Images of gels to be analysed are imported , opened in the software and labelled appropriately.
  • The cropping tool is used to select the region on the gel having maximum spot density and eclude the regions with out spots from analysis.
  • The spot picking tool selects the spots on the gel using certain user- defined criteria. The software then records the pixel intensities of the spots.
  • A 3-D graphical representation of the spots can be seen using the 3-D view tool.
  • Spot parameters such as volume , intensity , possible pI , molecular weight rtc. can be obtained through the software.
The resulting data can be compiled together to understand the global expression profile of the particular sample.
A schematic representation of the entire process of Global expression analysis is represented in Fig.7

 

Fig. 7: 2-D electrophoresis and analysis: A schematic representation of all processes involved in global expression analysis, starting from preparation of protein sample to analysis of 2-D gels

 

 

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