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                                 PROTEOMICS -TOOLS
AND DATABASES.

SYNOPSIS:

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·      
INTRODUCTION

·       NEED FOR PROTEOMICS STUDY

·      
PROTEOMICS
TOOLS AND DATABASES

                              
–Mass spectrometry

                              
–Protein microarray

    
–Two-dimensional gel electrophoresis

                              
–Reverse phase chromatography

                                –BLAST

                                 –FASTA

·      
APPLICATIONS
OF PROTEOMICS

·       LIMITATIONS OF PROTEOMICS

·       FUTURE INSIGHT IN PROTEOMICS.

·       CONCLUSION

·      
ARTICLE

 

 

 

 

INTRODUCTION:

 

                          The proteins are
macromolecules that are made up of amino acids. These proteins play major role
in the biological functions of all organisms like signal transduction, DNA
replication, catalysing metabolic reactions etc. The total amount of proteins
present at a particular period in a cell is called proteome. The diverse and large-scale
study of these proteins is called as proteomics. It
is estimated that there are around 50,000 to 2 million different proteins in
our biological system. Proteomics characterises them and determines their shape
and structure. The word proteomics was coined by Marc welkin in 1994. The first
proteomics lab was established in Macquarie university in 1995. Proteomics is
an interdisciplinary field that covers the emerging research fields and helps
us in gaining deeper insight on genomics, phylogenetics etc. knowledge on proteomics
helps in designing drugs, understanding complex structure and also using
proteins as biomarkers.

                              

 

 

WHAT MAKES STUDYING PROTEINS DIFFICULT?

The
branch of proteomics is similar to that of genomics which deals with the study
of the entire genes in an organism. But studying proteins is comparatively
difficult than studying genes due to the following reasons,

·       
The genome of an organism almost remains
constant but the proteome tends to differ from cell to
cell depending on the functional requirement and the physiology of the
cell.

·       
Quantifying the
amount of protein produced based on mRNA is also difficult because the quantity
depends on the number of genes expressed.

·       
Not all the proteins produced after
translation are functional. Some undergo post
translational modifications like methylation, acetylation,
phosphorylation, ubiquitination etc to become active. Thus, when the proteome is
estimated they may also be calculated.

·       
Some proteins are expressed
only during a particular period in an organism’s life time, example the
proteins involved during the developmental stages, carcinogenesis etc are
expressed only during those period, thus, making them difficult to study.

In
order to overcome these difficulties, the field of proteomics was combined with
another interdisciplinary field bioinformatics to study in detail about them.

 

 

TOOLS AND DATABASES USED IN PROTEOMICS:

 

                                           The
term bioinformatics
was coined by Paulien Hogeweg and Ben Hesper in 1970. Bioinformatics in biology
helps in sequencing the samples and organising the query data, comparing the
data and understanding then evolutionary aspects and simulating the models of
various compounds. Combining bioinformatics and proteomics made the study
easier and helped to unravel a lot of physiological functions.

The
various tools and databases that are used in proteomics are

·       
Mass spectrometry

·       
Protein microarray

·       
Immunoassays

·       
Two-dimensional gel electrophoresis

·       
Reverse phase chromatography

·       
BLAST

·       
FASTA

MASS SPECTROMETRY:

Mass
spectrophotometer is an instrument used in the quantification and quantifying a
large number of proteins

 

 

 

 Principle- mass
spectrometry coverts the liquid or solid-state protein into gas phase by
ionization and then accelerating them between electric or magnetic field so
that the deflection amplifies them and the readings are recorded.

 

 

 

The
ionisation of proteins can be done through electrospray ionisation (ESI) and
through matrix-assisted laser desorption/ionisation (MALDI)

·       
In electrospray ionisation the proteins in
solutions are converted into ions and ionise them. The advantage of using ESI
is that it retains most of the non-covalent interactions and produces more
amounts of charged ions.

·       
In matrix-associated laser
desorption/ionisation the proteins in solid form is embedded in matrix and are
ionised by creating pulse through laser light. The advantage of MALDI is that
it is fast and quantifies a large number of proteins.

PROCEDURE-

1.     
First the proteins are ionised using ESI
or MALDI technique

2.     
Then the proteins are enzymatically
digested using enzymes like pepsin, trypsin etc

3.     
The peptides are then introduced into mass
spectrometer and various analysis like peptide mass fingerprinting, tandem mass
spectrometry are performed

APPLICATIONS:

§  Through
peptide mass fingerprinting various proteins can be identified and t its
structure can also be determined.

§  The
sequence of a particular protein can be fed into the system and the peptide
sequence can be synthesised (De novo sequencing)

§  By
incorporating heavy isotopes like carbon-13 the protein can be quantified.

                                                      

PROTEIN MICROARRAY

                                In protein microarray the proteins are arrayed
onto a solid surface made of glass, silicon or nitrocellulose so that they are
made immobile and prevented from denaturation. When probes of our interest are
added to the array, the reaction between the proteins in the array and probe
will cause fluorescence which can be read by laser.

ASSAYS IN MICROARRAY CHIP

 

The
advantage of using protein microarray in proteomics is that it
is rapid, time consuming and requires only a minimal amount of sample. There
are 3 types of protein microarrays, they are

·       
Analytical microarray
– the proteins in this array are probed with other proteins so that their
binding reaction can provide results regarding the expression levels of those
proteins.

·       
Reverse phase protein microarray
– samples like cells in tissue lysate are used here. The cells are arrayed onto
the microarray and then probed with antibodies which can be detected using
chemiluminescent or fluorescent assays. This helps in detecting the defects in
post-translational modifications.

·       
Functional protein microarray –
they are constructed by immobilizing the entire set of proteins on the array.
They help in studying the interactions of proteins with DNA, RNA and other
proteins. The difference between functional protein microarray and analytical
microarray is that the former contains the full length functional proteins in
an array.

PROCEDURE
OF PROTEIN MICROARRAY

APPLICATIONS:

·       
Micro array plays a major role in
expressing the protein profiling. That is the functioning of the proteins in
the entire cell lysate can be identified.

·       
The interaction of the proteins with
various other molecules can also be easily deciphered.

The
greatest advantage of using protein microarray is that analysis of proteins can
be done in large scale and it is reproducible. But the challenges of using
microarray is that producing chips with long self-life is difficult, care must
be taken to avoid non-specific binding when adding probes, extracting the
detected protein from the chip is difficult. The cost for scanning the array is
also high.

 

 

TWO-DIMENSIONAL GEL
ELECTROPHROSIS:

The
two-dimensional gel electrophoresis is a technique used to separate and analyse
complex protein mixture. It was introduced in 1975 by O’Farrell and Klose. Here the proteins are
separated in the following major steps

                               
i.           
Initially in the first dimension the
proteins are separated according to their isoelectric point and it is called
isoelectric focusing (IEF)

                             
ii.           
In the second dimension the proteins are
separated according to their molecular weight in SDS-poly acrylamide gel
electrophoresis (SDS-PAGE).

                          
iii.           
After separating the proteins in the gel
stains like silver or Coomassie blue are added to visualize the proteins.

                           
iv.           
The proteins that are separated can be
eluted and can be further subjected to mass spectrometry for further analysis.

TWO-DIMENSIONAL GEL ELECTROPHROSIS PROCEDURE

 

 

GEL CONTAINING PROTEIN SAMPLES AND COOMASSIE BLUE DYE

 

TWO-DIMENSIONAL GEL ELECTROPHROSIS RESULT IN MASS SPECTROMETER

 

The
gel separates the proteins and the individual proteins can be seen as spots on
the gel. For further quantification of these proteins other 2D gel analysis
software likedelta2D, ImageMaster, Melanie etc are needed. The advantages of
using these software is that they can very clearly differentiate closely
located spots or overlapped spots and decipher their functions.

 

 

 

 REVERSE
PHASE CHROMATOGRAPHY:

Reverse
phase chromatography is a technique used to separate proteins based on their
solubility. This is a modification of the normal chromatographic technique
where the stationary phase packed with silica is modified with silyl ethers along
with non-polar alkyl groups. Thus, the stationary phase is hydrophobic. The
mobile phase contains polar organic solvents like isopropanol, methanol,
butanol etc. generally all the proteins will contain hydrophilic and
hydrophobic groups.  But proteins with
net hydrophobic group will precipitate in the hydrophobic stationary phase. By
increasing the concentration of the organic solvent, the precipitated protein
can be eluted.

There
are 4 main stages that are involved in reverse phase chromatography, they are

·       
Equilibration-  here hydrophobic column is equilibrated with
the sample buffer so that the water molecules remain in the junction between
the column and the buffer.

·       
Sample application-
here the sample protein is combined with higher molecular weight amino acids
and the applied on the column. This ensures that only the sample proteins bind
and the other proteins are removed.

·       
Elution-
now the hydrophobicity of the buffer is gradually changed and the bound
hydrophobic proteins are released.

·       
Regeneration-  the remaining proteins are washed off from
the stationary phase and a less hydrophobic environment is created.

Another
important chromatographic technique that is used in proteomics apart from
reverse-phase chromatography is ion-exchange chromatography. The ion-exchange
chromatography uses charged particles in their stationary phase. The charge depends
on the protein to be separated.

Generally,
in proteomics the cation-exchange
chromatography is generally used. Here the stationary phase is made up of
negative charge and hence it attracts positively charged proteins. The cation
exchangers can be weak or strong depending on the lose charge as pH is varied. The weak cation exchangers are the
carboxymethal group and the strong cation exchangers are sulfopropyl groups. 

BLAST

Another
important bioinformatics tool that is used in proteomics is Basic Local Search Alignment
Tool (BLAST). The BLAST program was developed by Stephen Altschu, Warren Gish, Webb
miller, Eugene Myers and David j. Lipman in 1990 at the national institute of
health. It helps in searching sequences. There are many methods of sequencing proteins
like Maxam and Gilbert method, Sanger’s enzymatic method etc. the sequenced
data are uploaded in the depository sites like national centre for
biotechnology information (NCBI), European molecular biology laboratory (EMBI).
The deposited sequence can be retrieved from these sites and are subjected to
BLAST analysis. Before the creation of BLAST database doing sequence search for
a particular protein or nucleotide was difficult.

In
order to search for the relation between sequences, the query sequence if
seeded and then BLAST makes local alignment trying to match the sequences and
establish the relation between them. The BLAST analysis has threshold values
and if the query sequence matches with the threshold then it indicates that the
alignment will be included in the results given by BLAST.

USES OF BLAST ANALYSIS:

·       
Using BLAST homologous species can be
identified. this will help in research in finding alternative samples.

·       
Through BLAST analysis, the phylogenetic
relationship can be established. the results provided by computational
phylogeny is less reliable and hence, BLAST is considered more useful.

·       
BLAST compares the chromosomal sequences
and it helps DNA mapping.

·       
BLAST can locate common genes among the
related species and utilise it in map annotation between organisms.

BLAST

 

BLAST RESULT FOR INSULIN A-chain

 

FASTA:

Like
BLAST, FASTA is also a DNA, protein sequence alignment package that is commonly
used in proteomics. FASTA was described by David J. Lipman and William R. Pearson
in 1985. The FASTA package contains programs for protein sequencing, nucleotide
sequencing etc. it uses Smith-Waterman algorithm in additional to heuristic
search method.  When the sequence for the
query nucleotide or amino acid is feeded into the FASTA database, it does local
alignment search tool and provides the similar sequences. It can also infer the
functional and evolutionary relationship between the sequences.

 

 

 

 

 

 

 

FASTA analysis

 

 

 

APPLICATIONS OF
PROTEOMICS:

Due
to the understanding in the field of molecular biology and developments made in
the field of proteomics and bioinformatics, a large benefit to man kind is
obtained. The various applications of proteomics are

·       
In the designing of drugs-  the structure of toxins secreted by bacteria
and other antigens can be deciphered through various tools used in proteomics.
Their epitopes can be analysed and suitable antibiotics and other drugs can be
designed to neutralise its effects.

·       
Protein biomarkers-
one that is becoming important in the drug developmental process is the need
for biomarkers. Biomarkers are important in identification of bacterial
antigens, pathogenic processes, indicators of normal biological processes and
they also help in understanding the pharmacological response to therapeutics.
Using proteomics individual biomarkers respective to particular proteins are
produced and the above-mentioned functions are elucidated. Examples- western
blot, enzyme linked immunosorbent assay (ELISA).

·       
Structural proteomics-
a large amounts of new genes are constantly being discovered and their
functions are yet to be known. In these conditions analysing the structure of
the protein helps to know about the binding ability and its interactions. the
structural proteomic tools like NMR spectrometry, X-ray crystallography help in
this.

·       
Proteogenomics –
many diseases are developing due to improper folding and functioning of
protein. They occur mainly due to the mistakes that happen during the post
translational modifications. thus, the parallel analysis on proteomics and
genomics helps us in identifying the defects in post translational
modifications and rectifying them.

·       
As genomics helps to understand the
genetic differences among individuals, proteomics helps in developing personalised
medicines that can be specifically effective for each individual.

Limitations of proteomics
study:

Though
proteomics study has many applications and advantages, there are few
limitations also. They are

·       
Generally, when the whole-body protein is
considered into study, the protein content may be affected due to the
degradation rate.

·       
Many proteins are found to be in
association with other proteins or RNA molecules and function only in the
presence of them. this makes quantifying them difficult.

·       
Many proteins undergo post translational
modifications like phosphorylation, methylation, deamination etc and only them
they become active. This makes analysing them difficult since, both
preproprotein and the active protein will be taken into account for quantification.

·       
Proteomics analysis are costly
particularly the scanning procedure in mass spectrometry, protein microarray
etc

·       
This procedure also demands trained and
experienced personals to carry out the procedures.

FURTHER INSIGHT IN
PROTEOMICS:

Since
researches are finding proteomics to be interesting they are finding ways and
methods to overcome the difficulties and draw backs that they faced in the
already existing techniques. Some of the future insights and developments
researches are developing are

ü  Advances
are being made in the quantitative proteomics.  Efforts are being made to understand the
biological mechanisms clearly and tools are being developed to quantify the
protein. These quantifications allow us to measure the cancer proteins and diagnose
them. This technology will be helpful in the fields like cancer biology, stem
cell research, evolutionary biology etc.

ü  Till
now proteomics has only concentrated in studying the cellular proteins. Efforts
are now being made to study the plasma proteins and it is the toughest proteome
study till now. The plasma contains proteins like cytokines, interleukins,
hormones, immunoglobulins etc. when these proteins are being studied it would
be easier to interpret diseased conditions and syndromes and they can be
rectified.

 

CONCLUSION:

 

Thus,
currently the field of proteomics is rapidly emerging and trying to find
solutions for all human illnesses. Since the corporate sector is also seeing
the development of biopharmaceuticals, the demand for the global proteomics
market is also increasing. Hence the future may see the development of
personalized medicine and drugs to cure diseases. Thus learning about proteomics
in additional to bioinformatics and molecular biology is becoming inevitable.

 

Article:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200191/

this
article says that of all the developing cancers one such important malignant
cancer is the pancreatic cancer. It may be caused due to a variety of reasons
and the defects of the current trending mode of medicine is that they find it
difficult to diagnose and then treat cancer. In such circumstance the
development in the field of proteomics is helping in formulating many tools and
standardising the biomarkers for pancreatic cancer and diagnosing it. further
proteomic research is also being carried out to find an effective treatment for
pancreatic cancer.

REFERENCES:

·       
 Geoffrey
M. Cooper, Robert E. Hausman; the cell, a molecular approach; Boston
university; ASM PRESS; 2012

·       
 Lodish
et.al; molecular cell biology; W. H. Freeman
and company, sixth edition.

·       
https://en.m.wikipedia.org/wiki/Proteomics

·       
https://www.ebi.ac.uk/training/online/course/proteomics-introduction-ebi-resources/what-proteomics

·       
/en.wikipedia.org/wiki/Protein_mass_spectrometry#/media/File:
Quantitative_mass_spectrometry.svg

·       
https://books.google.co.in/books?id=nI1RSmX0GXUC=PA210=PA210=https:/2D+gel+electrophrosis=bl=sdb9GT66QD=jcASmZtBpMLCQgvupnStfyGF8ho=en=X=0ahUKEwi3lPfG28rYAhUUS48KHZz0DbkQ6AEINDAC#v=onepage=https%3A%2F2D%20gel%20electrophrosis=false

·       
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200191/

 

 

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