NURSAH ASLAN

2092434

LABORATORY OF ADVANCED DNA, RNA AND PROTEIN ANALYSIS

PROTEOMICS LABORATORY REPORT
1. Introduction
Proteomics is a discipline that focuses on the wide analysis of proteins within a biological system. It ensures
worthy insights into the structure, modification, function and interactions of proteins. Proteomic approaches
are especially useful for biomarker identification that where proteins serves as markers for specific
conditions. In food technology, proteomics has a crucial role in the development of simple and fast methods
for analyzing complex or highly processed food matrices and quantification of trace levels of analytes with
high selectivity. Proteomics technology using different high-performance separation techniques such as
two-dimensional gel electrophoresis, one-dimensional and multidimensional chromatography, combined
with high-resolution mass spectrometry has the power to monitor the protein composition of foods and their
changes during the production process. The use of proteomics in food technology is presented, especially
for characterization and standardization of raw materials, process development, detection of batch-to-batch
variations and quality control of the final product. Further attention is paid to the aspects of food safety,
especially regarding biological and microbial safety and the use of genetically modified foods (D. GASO-
SOKA C et al.,2010)
In order to identify biomarkers according to their isoelectric point and molecular mass, 2DE gel
electrophoresis commonly employed. As an abstract to the workflow that will be mentioned below, the
main principle is to seperating proteins that are solubilized by isoelectric values with the use of IEF
(isoelectric focusing) and then by molecular mass with the use of SDS-PAGE. After protein separation and
digestion of proteins, the quantification and identification of proteins are done by using mass spectrometric
techniques (Gallardo et al., 2013) like LC‐MS, MALDI‐TOF MS, or MS/MS. MALDI‐TOF MS is,
however, used in protein identification while MS/MS or LC‐MS/MS can be used for identification,
quantification, as well as protein characterization.
Proteomic analysis can be very useful to provide both general and specific information on the “interaction
proteomes” used by hosts and pathogens and in the investigation of seafood toxins, the proteome of
Alexandrium tamarense, a paralytic shellfish toxin (PST)-producing dinoflagellate and determined
biomarkers of toxicity that were present in all toxic strains of this algae. 2DE and MS were used for
identification of these proteins. Further analysis by MALDI-TOF MS and N-terminal amino acid
sequencing revealed that they are isoforms of the same protein. Also, 2DE-based proteomic approach was
applied in order to discriminate toxic and nontoxic strains of the algae Alexandrium minutum and a minimal
variation in morphological features was found. The most notable differences between these strains were
several abundant proteins with isoelectric points ranging from 4.8 to 5.3 and apparent molecular masses
between 17.5 and 21.5 kDa (Giacometti et al., 2013)
Besides all of the advantages that mentioned above, due to limitations of the detection of low abundance
proteins,biases in sample preparation techniques that can lead incostistencies in the data, due to the
limitations in sensitivity or protein sequence coverage; some proteins may be missing. Also Proteins can
undergo various post-translational modifications (PTMs) and exist in different isoforms. Identifying and
characterizing these modifications and isoforms can be difficult Those type of occasions may count as
disadvantages or in those cases, it may require some additional techiques either.
2. Methods
a. Protein Solubilization and Denaturization
In order to perform lysis of the sample, lysis buffer which contains Urea, CHAPS, Triton, Tris-HCl and
protease inhibitor coctail has been used. Urea is a chaotropic agent that has good capacity to denature
proteins by breaking hydrogen bonds and hydrophobic interactions which gives folded structure to proteins.
And CHAPS is a non-ionic zwitterionic detergent which used to increase solubility of hydrophobic
proteins.The reason for us to choose a non-ionic detergent is, ionic detergents and salts may can interfere
with the separation that depends on the charge during the IEF (A.Görg et al., 2004). After lysis, we
collected supernatants through centrifuge. In order to eliminate excessive salts or other interfering
molecules and ensure compatibility for further processes such as IEF, we performed buffer exchange which
made by gel filtration chromotography using Bio-Spin Columns which are small columns that are ready to
 use for rapid and efficient cleanup and purification of pproteins and nucleic acids using a swinging bucket
centrifuge. At the end of this process, we collected the portion of interest from the upper side of the column.
b. Calorimetric Analysis
In order to determine the total protein, photometric protein determination is an important step in using the
relationship between concentration and light absorption. The Beer-Lambert law forms the basis for these
experiments, stating that the rate at which a particle absorbs light is directly proportional to its absorption
Different methods for photometric protein determination are available, each with specific principles and
applications.Direct absorbance analysis measures the absorbance of unmodified protein samples at 280 nm,
relying primarily on aromatic side chains of tryptophan and tyrosine residues but more complex assays,
such as the Bradford assay, have been performed using Coomassie Blue dye binding functions in
accordance with the present protein. The Lowry method combines Biuret reagent with Folin-Ciocalteu
reagent to measure the color change of the sample, which indicates protein concentration For photometric
protein determinations, standard solutions are prepared to construct a calibration curve, and the absorbance
values of the standards are measured. Once the curve is established, the calibration curve can be used to
measure the absorbance of unknown samples have been analyzed by the calibration curve to determine their
protein content These photometric protein identification methods have wide applications, such as
quantification of nucleic acids, characterization of biomimetic materials, and antibody detection (JoVE
Science Education Database). In our case, we used Bovine Serum Albumin (BSA) as a standard. We
diluted BSA and our sample by double-distilled water and for the calorimetric assay BCA and copper
solution mixed with diluted protein sample and BSA. And briefly, the Cu2+¿ ¿ reduced to Cu+¿¿ by the
proteins and then interacts with BCA and provides a purple color. In the context of the intensity of color
indicates the amount of protein. And we created a standard curve that is created by the known
concentrations of BSA.
c. Protein Separation
i. Isoelectric Focusing (IEF)
The first dimension of protein seperation is IEF with the use of IPG strips. IPG strips are immobilized pH
gradient strips that are used for the seperation of protein electrophoretically by pH range. IPG’s can be build
in different pH ranges between pH 2.5 and pH 12 as well as in different lenghts that derives from 7-24 cm. In
our case, we used 7cm long strips with a pH range of 4 to 7, which means that also we are losing proteins that
are outside of this pH range. Prior to IEF, we rehydrated the IPG dry strips by the use of rehydration buffer
that contains urea, CHAPS (non-ionic detergent), Bromophenol blue and DTT that added priorly. DTT is a
reducing agent which interferes with disulphide bridge formation in proteins and by that, it causes
denaturation. It is important to be sure if all the proteins are denatured before further processes. And
bromephenol blue dye is used to see the movement of proteins through gel. The method that has been
employed for the rehydration was sample-in-gel rehydration (A.Görg et al., 2004). The solution and sample
pipetted into the equilibration tray and IPG strips are placed on top with gel side facing down. After then, we
covered the strips with mineral oil in order to prevent dehydration of strip. We let them sit overnight at the
bench to be sure the gel absored proteins. The day after, we took the strips from the tray and drained the
mineral oil. Then we placed the strips into the channels of focusing tray with wires that are covered with wet
absorbant papers. Then, focusing tray placed on IEF and electrophoresis carried out in order to seperate
proteins.
ii. Equilibration of IPG gel strips
In order to be able to perform second dimension, the important part is to equiibriate the IPG strips to provide
a proper protein interaction with SDS (sodium dodecyl sulfate). This step is crucial due to the proteins bound
to IPG gel matrix stronger than other components. It is handled by the use of equilibration buffer which
contains urea and glycerol, to minimize electroendosmotic efect which refers to the movement of liquid
induced by an electric field within charged matrix impacting the migration of analytes during electrophoresis,
Tris-HCl, SDS, DTT and urea. It facilitates the transfer of proteins from first dimension gel to second
dimension SDS-PAGE gel. After the equilibration for 15 minutes, we used the same buffer but instead of
DTT, replaced with IAA(iodoacetamide) which serves to alkylate any remaining DTT (A.Görg et al., 2004).

iii. SDS-PAGE
SDS-PAGE is the second dimension for protein seperation. It is so far the most employed technique for
quantitative expression profiling of protein mixtures including cell lysates and it allows for the seperation of
proteins based on their pI, molecular mass, solubility etc. Gel has been prepared by bisacrylamide and
acrylamide and polymerased with TEMED which is the polymerizing agent. Proteins are denatured and by
the addition of sodium dodecyl sulfate and mixed with loading solution that has bromophenol blue dye. It
acquire a net negative charge proportional to their length. The gel has been subjected to an electric field
which allows to the migration of proteins. Smaller proteins are migrated and moved further on gel. The strip
that is obtained before, putted on top of the SDS-PAGE with the use of agarose and bromophenol mix,
stabilized. And the system connected to the power source and runned.
3. Further Analyses
a. Gel Staining
In order to prevent protein diffusion, the proteins that are seperated acquires fixation. We
achieved fixation by ethanol and acetic acid. Those fixatives helps to maintain the
position of the proteins and prevents passive diffusion overtime. Protein visualization is
performed by Coomassie staining and Silver staining. Coomassie dye binds to the
proteins that are fixed and forms blue color and in order to remove excess dye on gel
double distilled water and destaining solution has been used. At the end, the intesity of
the color gives information about relative abundance but since we need more sensitive
techniques, we performed silver staining. It is a sensitive technique for protein
visualization. We placed the gel into silver nitrate solution. By that, silver ions binds to
amino side chains, particularly sulfhydryl and carboxyl groups of proteins. Aterwards,
we used developing solution which contains formaldehyde and sodium carbonate and by
that the silver-protein complex that we obtained as a result of the binding of silver ions to
proteins are reduced to metalic silver and the reduction occurs at the sites of protein spots
on the gel and it leads to visible silver deposits that makes protein spots/bands visible. At
the end, in order to stop the reaction, we used stopping solution which includes acetic
acid. By that, we prevent further silve accumulation and stabilized already developed
bands. Then we used fixing solution to preserve the integritiy of the bands and diffusion.
b. Immunobased Assays
i. Western Blotting
Western blotting is also known as immunoblotting and it is commonly used to
detect specific proteins in a mixture of proteins. The main principle behind is
antigen-antibody interactions. After the protein seperation has been done, we used
the gel that we obtained through electrophoresis are hydrated through water to
prevent drying. In order to transfer proteins on the gel to a nitrocellulose paper we
prepared a sandwich which consists of nitrocellulose paper, sponge, absorbant
paper. After we prepared the sandwich and clipped all together and placed it in
electrophoresis chamber.Then we added running buffer to chamber and ice to
prevent over heating and run it. After the transfer has been achieved, the membrane
is then incubated with labels antibodies specific to the protein of interest. The
unbound antibody is washed off leaving only the bound antibody to the protein of
interest.The bound antibodies are then detected by developing the film (Mahmood T
et.al, 2012). In order to visualize proteins, reversible Panceau S staining has been
used.

4. Biomarker Identification
In proteomics, protein discovery and characterization rely on mass spectrometry (MS) techniques. In a
suitable procedure, proteins are separated and analyzed by MS. These data are compared with sequence data
using Mascot and other search engines to identify proteins. In peptide mass fingerprinting (PMF), unknown
proteins are extracted and digested into peptides and analyzed by MS to identify proteins. Peptide
fragmentation fingerprinting (PFF) Tandem MS (MS/MS) for fragment ion data detects the protein . In both
 methods, the protein sequence must be present in the database. Post-translational modifications (PTMs) are
critical for protein function. MS characterizes PTMs based on significant changes in modified amino acids.
However, PTMs are often present in such low abundance that amplification methods are used. MS ionized
analytes and seperates them based on their mass to charge ratio using mass analyzers. The ion current is
detected and producing a mass spectrum (J. Gallardo, et al, 2013).
5. Results and Discussion

Figure 1 2DE gel silver staining

Figure 2 1D gel coomassie blue staining

Figure 1 represents the 1D gel results for peptides that

are obtained from various samples. Our sample is in
5th column (HeLa Mg132).
Figure 2 is the 2DE gel treated with silver staining. We
can say that the proteins that are close to left side is
more closer to ph 4 and reverse is closer to 7 because
of the ph interval of IPG strip. And for the upper side
of the gel we can say that proteins that are high in
molecular mass; for lower side situation is reverse.
Figure 3 represents the solubilized proteins that are
treated with BCA and the color represents the protein
concentration. And the wells that has water acts as
blank for spectrophotometric analysis.
Figure 3 Proteins that are treated with BCA solution

6. References
 Gallardo, J. M., Ortea, I., & Carrera, M. (2013). Proteomics and its applications for food authentication and food-technology research. TrAC - Trends
in Analytical Chemistry, 52(December), 135–141.
 Gašo-Sokač D, Kovač S, Josić D. Application of Proteomics in Food Technology and Food Biotechnology: Process Development, Quality Control and
Product Safety. Food Technology & Biotechnology [Internet]. 2010 Jul [cited 2023 Jul 3];48(3):284–95.
 Giacometti, Jasminka, Alena Buretić Tomljanović, and Djuro Josić. "Application of proteomics and metabolomics for investigation of food
toxins." Food research international 54.1 (2013): 1042-1051.
 Görg, A., Weiss, W., Dunn, M. J. (2004). Current Two-dimensional Electrophoresis Technology For Proteomics. Proteomics, 12(4), 3665-3685.
https://doi.org/10.1002/pmic.200401031
 JoVE Science Education Database. <em>Biochimica.</em> Photometric Protein Determination. JoVE, Cambridge, MA, (2023).
 Mahmood, T., Yang, P. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North Am J Med Sci, 9(4), 429.
https://doi.org/10.4103/1947-2714.100998