Experiment-1:
Global proteomic analysis
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 in the sample is measured.

Rehydration of IPG strips: Measured amount of sample is loaded onto the IPG strips.

IEF: This is the first dimension wherein sample proteins are separated on the IPG strips
on the basis of their isoelectric point.

Equilibration of the strips: The IPG strips are saturated with the 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
stimulations, 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 twodimensional (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 chose such that it is compatible 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 reswell tray. IPG strip
is placed over the sample such that the gel side is in touch with the sample. Cover fluid is then
added to the well 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. 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 2Iodoacetamide (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 Trisglycine 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 Van der 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 2D 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 analyzed are imported, opened in
the software and labeled appropriately.

The cropping tool is used to select the region on the gel having maximum spot density
and exclude the regions without 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 etc. 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 gel electrophoresis and analysis: A schematic representation of all the processes
involved in global expression analysis, starting from preparation of protein sample to analysis of
2-D gels is shown.