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A Short Review on Proteomics and its Applications

Article  in  International Letters of Natural Sciences · June 2014

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International Letters of Natural Sciences

12(1) (2014) 77-84 ISSN 2300-9675

A Short Review on Proteomics and its Applications

K. Chandrasekhar1, A. Dileep2, D. Ester Lebonah1, J. Pramoda Kumari1,*

1
Research Scholar, Department of Microbiology, S.V. University, Tirupati,
Andhra Pradesh, India
*Fax No: +91-0877-2289555
2
Junior Research Fellow, Department of Microbiology, S.V. University, Tirupati,
Andhra Pradesh, India
*E-mail address: [email protected]

ABSTRACT
Proteomics is the large scale of study of proteins, particularly their function and structure.
Proteomics is an excellent approach for studying changes in metabolism in response to different stress
conditions. In the present review focused on different types of techniques for the analysis of expressed
proteins. The techniques includes 2-D gel electrophoresis, MALDI-TOF/MS etc., play a vital role for
the analysis of novel proteins and their role in disease maintenance and treatment. The review also
concentrated on applicative perspective of proteomics in the fields of biomedical, agriculture and
food.

Keywords: Proteomics; 2-D electrophoresis; MALDI-TOF/MS; Matrix; Applications

1. INTRODUCTION

Protein, highly complex substance that is present in all living organisms. Proteins are
the polymers of amino acids. Emil Fischer and Franz Hofmeister, reported about proteins in
1902, Proteins play an important role in metabolic activities. Primary structure of protein is
determined by the sequence of specific amino acids, encoded by the mRNA, which directs
the proper folding of the polypeptide chain into the secondary structure. One type of
secondary structure is the alpha helix, a region of the polypeptide that folds into a corkscrew
shape. Beta strands are linear structures of polypeptides, bonding together to form a flat beta
sheet. Turns and coils interact chemically with each other to form the unique three
dimensional shape of the proper three dimensional structure creates the final protein. Many
proteins, however, have several different polypeptide subunits that make the final active
protein. For these proteins, the interactions between the different subunits form the
quaternary structure. One of the most promising developments to come from the study of
human genes and proteins has been the identification of potential new drugs for the
treatments of disease. This relies on genome and proteome information to identify proteins
 International Letters of Natural Sciences 12(1) (2014) 77-84

associated with a disease. The term “proteomics” was first coined in 1995 and was defined as
the large-scale characterization of the entire protein complement of a cell line, tissue, or
organism1,11,12. Proteomics is the large-scale study of proteins particularly their composition,
structures, functions, and interactions of the proteins directing the activities of cell 2,3. The
main theme of interest proteomics it gives a much better understanding of an organism than
genomics. Genomics can give a rough estimation of expression of a protein. Most of the
proteins function in collaboration with other proteins, and the main goal of proteomics are to
identify which proteins interact. After genomics, proteomics is often considered as the
advanced step in the study of biological systems. It is much more complicated than genomics,
mostly because while an organism’s genome is more or less constant, the total protein
expression profile always changes with time, micro and macro environmental conditions.
Mass spectrometry (MS) has been widely used in forensic science in the identification
of compounds, particularly illicit drugs. MS is a technique that allows the detection of
compounds by separating ions by their unique mass (mass-to-charge ratios) using a mass
spectrometer. The method relies on the fact that every compound has a unique fragmentation
pattern (mass spectrum). The sample is ionized; the sample ions are separated based on their
differing masses and relative abundance.

2. TYPES OF PROTEOMICS

Based on the protein response under stress conditions proteomics are classified into
different groups.

2. 1. Expression proteomics
Expression proteomics is used to study the qualitative and quantitative expression of
total proteins under two different conditions. Like the normal cell and treated or diseased cell
can be compared to understand the protein that is responsible for the stress or diseased state
or the protein that is expressed due to disease. Typically, expression proteomics studies are
addressed to the investigation of the expression protein patterns in abnormal cells. Ex.
Compare tumor tissue sample and the normal tissue can be analyzed for differential protein
expression. 2-D gel electrophoresis, mass spectrometry technique were used to observed the
protein expressional changes, which is present and absent in tumor tissue, when compared
with normal tissue. Which are over expressed and under expressed can be identified and
characterized protein activities multi-protein complexes, and signalling pathways4,5.
Identification of these proteins will give valuable information about molecular biology of
tumor formation and disease-specific manner for use as diagnostic markers or therapeutic
targets6.

2. 2. Structural proteomics
Structural proteomics helps to understand three dimensional shape and structural
complexities of functional proteins. Structural prediction of a protein when its amino acid
sequence is determined directly by sequencing or from the gene with a method called
homology modelling. Structural proteomics can give detailed information about the structure
and function of protein complexes present in a specific cellular organelle. It is possible to
identify all the proteins present in a complex system such as membranes, ribosomes, and cell
organelles and to characterise all the protein interactions that can be possible between these

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proteins and protein complexes. Different technologies such as X-ray crystallography and
NMR spectroscopy were mainly used for structure determination 7.

2. 3. Functional proteomics
Functional proteomics explains understanding the protein functions as well as
unrevealing molecular mechanisms within the cell then depend on the identification of the
interacting protein partners. The association of an unknown protein with partners belonging
to a specific protein complex involved in a particular mechanism would in fact, be strongly
suggestive of its biological function 8,9. Furthermore detailed description of the cellular
signalling pathways might greatly benefit from the elucidation of protein- protein interactions
in-vivo 10.

2. 4. Techniques involved in proteomics

In proteomic analysis both analytical and bio-informatics tools were used to
characterize protein structure and functions. Analytical techniques 2-D gel electrophoresis,
MALDI-TOF-MS were used. In case of bio-informatics numbers of software tools were used.

2. 5. 2-D gel electrophoresis

In 2-D gel electrophoresis, protein samples are resolved based on charge, in a step
called isoelectric focusing, and then based on molecular weight in second step 13. The result
is an image in thousands of small spots, each representing a protein. A good 2-D gel can
resolve one thousand to two thousand protein spots, which appear after staining, as dots in the
gel. 2-D gel electrophoresis technique is mainly used to compare two similar samples to find
specific protein differences.

2. 6. 2-D Electrophoresis workflow chart

Sample preparation

First Dimensional separation- IEF

Second Dimensional separation- Based on Molecular Weight

Detection

Image acquisition and analysis

Protein Excision, Digestion and Identification

Fig. 1. 2-D Electrophoresis workflow chart.

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Prepare the protein at a concentration and in a solution suitable for IEF. Choose a
method that maintains the native charge, solubility, and relative abundance of proteins of
interest. Separate proteins according to pI by IEF. Select the appropriate IPG strip length and
pH gradient for the desired resolution and sample load. Select appropriate sample loading and
separation conditions. Separate proteins according to size by SDS-PAGE. Select the
appropriate gel size and composition and separation conditions. Visualize proteins using
either a total protein stain or fluorescent protein tags. Select a staining technique that matches
sensitivity requirements and available imaging equipment. Capture digital images of the 2-D
patterns using appropriate imaging equipment and software. Then analyze the patterns using
2-D software. Excise protein spots of interest from the gel digest the proteins, and the digests
by MS.

2. 7. MS analysis
Mass spectrometry is an analytical technique that produces spectra of the masses of the
atoms or molecules comprising a sample of material. The spectra are used to determine the
elemental or isotopic signature of a sample, the masses of particles and of molecules, and to
elucidate the chemical structures of molecules, such as peptides and other chemical
compounds. Mass spectrometry works by ionizing chemical compounds to generate charged
molecules or molecule fragments and measuring their mass to charge ratios14. MALDI-TOF
is the most useful technique for protein identification.

2. 8. MALDI-TOF-MS
Matrix Assisted Laser Desorption/Ionisation is a soft ionization technique used in
spectrometry, allowing to analysis the biomolecules like DNA, protein, peptides.
Biomolecules and synthetic polymers have low volatility and are thermally unstable, which
has limited the use of MS as a means of characterization. These problems have been
minimized through the development of MALDI-TOF MS, which allows for the mass
determination of biomolecules by ionization and vaporization without degradation, a Laser
beam used to ionize the sample15.

Fig. 2. MALDI-TOF –MS analysis representing image.

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Protein sample have been characterized by HPLC or SDS PAGE by generating peptide
maps. These peptide maps have been used as fingerprints of protein or as a tool to know the
purity of a known protein in a known sample. Mass spectrometry gives a peptide map when
proteins are digested with proteolytic enzymes like trypsin. This peptide map can be used to
search a sequence database to find a good match from the existing database.

2. 9. Sample preparation
MALDI-TOF MS is used to characterise, biomolecules like proteins, peptides and
polymers of organic compounds. Sample preparation for MALDI-TOF is very interesting and
important step. Purify the protein sample before going MALDI-TOF analysis because it is
more tolerant to sample contaminants but contaminants can seriously disturb incorporation of
sample molecules with growing matrix crystals. Sample can mix with matrix in 1:2 ratio.
Different types of matrices are used based on sample, some of matrix are 2-(4-hydroxy
phenylazo benzoic acid, 2,4,6-trihydroxyacetophenone, 3-aminoquinolone, cinnamic acid,
etc.). Dried droplet technique is predominantly applied for MALDI-TOF analysis, protein
sample, mixed with matrix on a metal plate. The combination of matrices yielded slightly
performance16. Small volumes should be used for standard metal plates. On the other hand,
hydrophilic sample anchors are efficient for the generation of small spots17.

2. 10. Matrix
A good matrix consist the following properties i.e. Matrix must be able to absorb U.V
wavelength of usually 237 nm, being easily exited and ability to transfer of proton to the
sample molecules. Main role of the matrix is adsorption of energy from laser pulse, and then
transfer to sample this energy can causes the vaporisation of the sample. For protein samples
typical MALDI matrix consist of hydroxylated benzoic acid and cinnamic acid derivatives.

3. ADVANCED METHODS IN PROTEOMICS

3. 1. Isotope-coded affinity tags (ICAT)
It is a gel- free method for quantitative proteomics that relies on chemical labelling
reagents. These chemical probes consist of three general elements i.e. defined amino acid side
chain, an isotopically coded linker, and a tag for the affinity isolation of labelled
proteins/peptides. For quantitative comparison of two proteomes, sample labelled with
isotopically light, and other one is heavy version. Both samples combined with isotope-coded
tagging reagents. These peptides are analyzed by LC-MS. Tags were deuterium, 13C 18. The
technique mainly used the relative quantification of proteins present in two or more
biological samples. Visible isotope-coded affinity tags are the additional method in ICAT-
Visible tag that allows the electrophoresis position of tagged peptides to be easily monitored.

3. 2. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)

Isobaric tags for relative and absolute quantitation (iTRAQ), it is also a non- gel- based
technique used to quantify proteins. iTRAQ is used in proteomics to study quantitative
changes in the proteome19. Based on the covalent labelling of the N-terminus and side chain
amines of peptides from protein digestions with tags of varying mass, 4-plex and 8-plex are
the reagents can be used to label all peptides from different samples. The samples can be

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analysed by using mass spectrometry MS/MS. Different types of software’s are available for
analyse the MS/MS spectra’s i.e. j-Tracker, j-TraqX 20.

3. 3. Absolute Quantification (AQUA)

AQUA, studies the absolute quantification of proteins and their modification sates.
Covalent modifications can be used to prepare synthetic proteins. These modifications are
chemically identical to naturally occurring posttranslational modifications. These types of
peptides used to quantify the post translational modified proteins after proteolysis with the
help of tandem mass spectrometer.

3. 4. ESI-Q-IT-MS
Micro electrospray ionization (ESI)-Quadrupole ion trap (QIT) Time of flight (TOF)
mass spectrometer (MS) has a very good resolution. In ESI ionisation proteins are ionised in
solution and carry multiple charge state. The advantage of using ESI-QTOF analysis for
protein mass determination is that due to the high charge state of proteins their m/z
measurements is typically less than 2000 and the TOF detector has a very good mass
accuracy in this scan range. This result is more accurate mass measurements for proteins in
ESI-QTOF.

3. 5. SELDI-TOF-MS
The technique Surface-enhanced laser desorption/ionization (SELDI) is used for the
analysis of protein mixtures, it is an ionization method in mass spectrometry21. SELDI is
typically used with time-of-flight mass spectrometers and is used to detect proteins in clinical
samples; to compare protein levels with and without a disease can be used for biomarker
discovery22.

3. 6. Applications of proteomics
Proteomics is widely used technique in biological fields, mainly applied in Oncology
(Tumor biology), Bio-medicine, Agriculture and Food Microbiology.

3. 7. Oncology
Oncology refers study of Tumor cell, Tumor metastasis, is the process spread of cancer
from one organ to another non-adjacent organ cause death in patients24. The major challenge
in medicine to describe the molecular and cellular mechanisms underlying tumor metastasis.
Analyse the protein expressions correlated to the metastatic process which help to understand
the mechanism of metastasis and thus facilitate the development of strategies for the
therapeutic interventions and clinical management of cancer. Proteomics is a systematic
research, the main aim of this research is to characterize the protein expressions, functions of
tumor cells and widely used in biomarker discovery.

3. 8. Bio-medical applications
The study of interactions between microbial pathogens and their hosts is called
“infectomics”. It is very interesting area in proteomics. It deals with the fundamentals of the
infections origin and their effect on organs. The main aim of this research is to prevent or
cure disease at starting level. Advanced diagnostic issues related to emerging infections,
increasing of fastidious bacteria, and generation of patient- tailored phenotypes24.

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 International Letters of Natural Sciences 12(1) (2014) 77-84

3. 9. Agricultural applications
The applications of plant proteomics scientific research is still in budding stage.
Proteomics is also used to know plant-insect interactions that help identify candidate genes
involved in the defensive response of plants to herbivore 23. Population growth and effect of
global climate changes imposing severe limits on the sustainability of agricultural crop
production.

3. 10. Food Microbiology

The use of proteomics in food technology is presented especially for characterisation
and standardisation of raw materials, process development, and 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 foods25.

4. CONCLUSION

Based on the above findings the present review was concluded that the applications for
proteomics are relevant to all of the biological process and provides a means to utilise the
expressed protein data in a more effective way.

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( Received 30 May 2014; accepted 05 June 2014 )

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