General Principle and application of Proteomics

PROTEOMICS
SUBMITTED TO: SUBMITTED BY:
Dr . Smita Jain Taufik Ansari
(Assistant Professor)
(1st
Semester)
Central University Of Rajasthan
School Of Chemical Science And Pharmacy
Department of Pharmacy
M. Pharma
(Pharmacology)

CONTENT
 Introduction
 Role of Proteomics
 What is proteomics?
 Why Proteomics?
 Type of Proteomics
 Technique of Proteomics
 Application of Proteomics
 Role of Proteomics
 Conclusion
 Reference

INTRODUCTION
PROTEOMICS
The “proteome” can be defined as the overall protein content of a cell that is
characterized with regard to their localization, interactions, post-translational
modifications and turnover, at a particular time. The term “proteomics” was
first used by Marc Wilkins in 1996 to denote the Protein complement of a
genome.
Proteomics is crucial for early disease diagnosis, prognosis and to monitor the
disease development. Furthermore, it also has a vital role in drug development
as target molecules. Proteomics is the characterization of proteome, including
expression, structure, functions, interactions and modifications of proteins at
any stage.
Proteomics is one of the most significant methodology to comprehend the gene
function although, it is much more complex compared with genomic.

To study the dynamic protein products of
the genome and their interaction.
A large scale characterization and
functional analysis of proteins
expressed by genome.
PROTEIN
Genomics
Transcriptomics RNA
PROTEOMICS
DNA (gene)
Proteomics

PROTEOMICS
Proteome
Mining
P
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Functional
Proteomics
P
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M
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Structural
Proteom
ics
P
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Q
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Protein
Networks
3D
Interacto
me
Interactio
n Profiling
Functional
Characteriz
ation
Protein
Engineeri
ng
Disease
Mutations
Interactio
ns of
Proteins
Localizati
on of Prot
eins
Function
of
Proteins
Drug
Discovery
Protein
Complexe
s
3D
Structures
Target
Identificat
ion
Protein
Profiling
Protein
Identificat
ion
Expression
Profiling
Biomarker
Detection
Disease
Diagnosis
Role of
Proteomics

What is Proteomics
 Proteome indicates the total proteins expressed by a genome in a cell
or tissue.
 Biomarkers detection might allow identification of patients who
would benefit from further evaluation.
 With the development of proteomic techniques, proteome analysis
provides a fast, non-invasive diagnostic tool for patients with various
diseases.
 The advent of highly sensitive proteomic technologies can identify
proteins associated with development of diseases well before any
clinically identifiable alteration.
This Photo by Unknown Author is licensed under CC BY-SA-NC

Why Proteomics
Genomic
• DNA tell what
possibly
Transcriptomics
• RNA what
probably
Proteomics
• Proteins what's
actually happens

Why Proteomics
 The behavior of gene products is difficult or impossible to predict from gene sequence.
 Even if gene is transcribed , its expression may be regulated at the level of translation.
RNA
DNA mRNA Protein
Translation
processing
transcription
Proteolysis
Post
translational
modification
Compartmentations

Types of Proteomics
 Structural Proteomics:- The ultimate aim of this proteomics is to build a body of structural information that
will help predict the probable structure and potential function for almost any protein from knowledge of its
coding sequence.
 Functional proteomics:- It refers to the use of proteomics techniques to analyze the characteristics of
molecular protein-networks involved in a living cell.
 Expression proteomics:- It refers to the quantitative study of protein expression between sample differing
by some variable.

Technique of Proteomics
Figure : An overview of proteomics techniques.
https://doi.org/10.1093/chromsci/bmw167

 Conventional techniques:
 Chromatography-based techniques
 Ion exchange chromatography:
o The IEC is a versatile tool for the purification of proteins on the basis of charged groups on its surface.
o The proteins vary from each other in their amino-acid sequence.
o The net charged contain by a protein at physiological pH is evaluated by equilibrium between these charges. Initially, it
separates the protein on the basis of their charge nature (anionic and cationic), further on the basis of comparative charge
strength.
o The IEC is highly valuable due to its low cost and its capacity to persist in buffer conditions .
 Size exclusion chromatography:
SEC separates the proteins through a porous carrier matrix with distinct pore size on the basis of permeation;
therefore, the proteins are separated on the basis of molecular size.

Continue….
 Affinity chromatography:
o The affinity chromatography was a major breakthrough in protein purification that enables the researcher to
explore protein degradation, post-translational modifications and protein–protein interaction.
o The basic principle behind the affinity chromatography is the reversible interaction between the affinity
ligand of chromatographic matrix and the proteins to be purified .
o The affinity chromatography has a wide range of applications in identification of microbial enzymes
principally involved in the pathogenesis.

 Enzyme-linked immunosorbent assay:
o In 1971, Engvall and Pearlmann published the first paper on ELISA and quantified the IgG in rabbit
serum using the enzyme alkaline phosphatase.
o The ELISA is highly sensitive immunoassay and widely used for diagnostic purpose.
o The assay utilizes the antigen or antibodies on the solid surface and addition of enzyme-conjugated
antibodies to and measure the fluctuations in enzyme activities that are proportional to antibody and
antigen concentration in the biological specimen.
This Photo by Unknown Author is licensed under CC BY

 Western blotting:
Western blotting is an important and powerful technique for detection of low abundance
proteins that involve the separation of proteins using electrophoresis, transfer onto nitrocellulose membrane and
the precise detection of a target protein by enzyme-conjugated antibodies.

 Two-dimensional gel electrophoresis:
 The two-dimensional polyacrylamide gel electrophoreses (2D-PAGE) is an efficient and reliable method for
separation of proteins on the basis of their mass and charge.
 2D-PAGE is capable of resolving ~5,000 different proteins successively, depending on the size of gel.
 The proteins are separated by charge in the first dimension while in second dimension separated on the basis
of differences between their mass.
 The 2-DE is successfully applied for the characterization of post-translational modifications, mutant proteins
and evaluation of metabolic pathways.
 The membrane proteins from the cell wall of Listeria innocua and Listeria monocytogenes involved in the
host–pathogen interactions were analyzed with 2-DE and 30 different proteins of two strains were identified .
 This approach was useful for the comparative study of exotoxins and virulence factors released by
enterotoxigenic strains of two food-derived Staphylococcus aureus strains.

General Principle and application of Proteomics

 X-ray crystallography:
o X-ray crystallography is the most preferred technique for three-dimensional structure determination of
proteins.
o The highly purified crystallized samples are exposed to X-rays and the subsequent diffraction patterns are
processed to produce information about the size of the repeating unit that forms the crystal and crystal
packing symmetry.
o X-ray crystallography has an extensive range of applications to study the virus system, protein–nucleic acid
complexes and immune complexes.
o Further, the three-dimensional protein structure provides detailed information about the elucidation of
enzyme mechanism, drug designing, site-directed mutagenesis and protein–ligand interaction .

 Mass spectrometry:
o MS is used to measure the mass to charge ratio (m/z), therefore helpful to determine the molecular weight
of proteins.
o The overall process comprises three steps.
o The molecules must be transformed to gas-phase ions in the first step, which poses a challenge for
biomolecules in a liquid or solid phase.
o The second step involves the separation of ions on the basis of m/z values in the presence of electric
or magnetic fields in a compartment known as mass analyzer.
o Finally, the separated ions and the amount of each species with a particular m/z value are measured.
o Commonly used ionization method comprises matrix-assisted laser desorption ionization (MALDI),
surface enhanced laser desorption/Ionization (SELDI) and electrospray ionization (ESI)

This Photo by Unknown Author is licensed under CC BY
Figure: Mass spectrometry technique

Application of Proteomics
o Protein sample identification/confirmation.
o Protein sample purity determination.
o Detection of post-translational modifications
o Detection of amino acids substitution.
o Mass fingerprint identification of proteins.
o Nutrition Research
o To identify unknown protein of intrest.
o Quantify protein and peptide.
o Protein Biomarker.

Conclusion
In the previous several years, tremendously useful advances are made in the field of proteomics. The
technologies are rapid, sensitive and provide greater proteome coverage. Furthermore, combination of these
technologies has achieved success in purification, analysis, characterization, quantification, sequence and
structural analysis and bioinformatics analysis of large number of proteins in all types of eukaryotic and
prokaryotic organisms. All fields related to biological sciences have been benefited with increasing use of
proteomics techniques. However, further work is still required to improve the reproducibility and
performance of well-known proteomics tools.

Reference
 Cristea, I.M., Gaskell, S.J., Whetton, A.D.; Proteomics techniques and their application to
hematology; Blood,(2004); 103(10): 3624–3634.
 Wilkins, M.R., Sanchez, J.-C., Gooley, A.A., Appel, R.D., Humphery-Smith, I., Hochstrasser, D.F., et
al. .; Progress with proteome projects: why all proteins expressed by a genome should be identified and
how to do it; Biotechnology and Genetic Engineering Reviews, (1996); 13(1): 19–50.
 Canales, R.D., Luo, Y., Willey, J.C., Austermiller, B., Barbacioru, C.C., Boysen, C., et al. .; Evaluation
of DNA microarray results with quantitative gene expression platforms; Nature Biotechnology,
(2006); 24(9): 1115–1122.

General Principle and application of Proteomics

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