PROTEOMICS

Presenter-Dr. Shashikala C

Moderator-Dr.Kala

Black swallow tail- larvae and butterfly

Introduction The terms proteome and proteomics were coined by Mark wilkins and

colleagues in the early 1990’s.

.

Definition

That’s just not a protein biochemistry !

Proteome :

is the complement protein found in a single cell ina particular environment./ is complete collection ofproteins encoded by genome of an organism.

Proteomics :

is the study of composition, structure, function andinteraction of the proteins directing the activities of eachliving cell

Types of proteomics

• Interaction proteomics-

protein-protein association

• Expression proteomics-

protein quantification

Life cycle of a protein

The level of any protein in the cell at any given time is controlled by

1. Rate of transcription of the gene

2. The efficiency of translation of m RNA into protein

3. The rate of degradation of protein in the cell

Protein structurePrimary structure- is sequence of specific amino acid

Secondary structure- the primary polypeptide chain gets properly folded

In the form of alpha-helix ,bet a pleated sheet, random coils and turns

Tertiary structure: secondary structure interact with each other

chemically to form the 3 dimensional shape of the proteins.

Quaternary structure: interaction between different polypeptide unit

Domains : discrete portions of the proteins that fold independently

from the rest of protein and they have their own function and serve as

one of the building blocks of that proteins.

Motif : domains contain a region of conserved pattern of amino acids or

a conserved combinations of the structural elements formed

by folding of near by amino acid sequences.

Determining the protein structure/ polypeptide sequence by:

1. x-ray crystallography

2. Nuclear magnetic resonance

3. Protein predicting programmes- computer based

Relationship between structure and function

• Hydrophobicity is determined by primary and secondary structure

Eg. - Membrane spanning regions of membrane proteins are typically alpha helices made of hydrophobic AA which interact with hydrophobic lipids forming stable membrane structure

- Hb is soluble protein found in cytoplasm of RBC’s as single

molecules. In sickle cell anemia mutation in beta globin chain

protein increases its Hydrophobicity and protein molecules stick to

each other to avoid aqueous environment.

- folding of amino acids, in a primary sequence, which are distant from each other forms active site of an enzyme and ligand binding site of a receptor

Post translational modifications• Glycosylation-structural components of cell surface helps in binding

by the receptors and eliciting immune response.

eg. ABO blood group antigen, Human IgG

• Phosphorylation – play a role in the regulation of many cell processes such as growth and cell cycle control.

• Sulfation

Genomics and ProteomicsThe genome sequencing project of late 1990’s yielded complete genome

sequence of bacteria, yeast, nematodes, drosophila and also complete

sequence of human genome . while expression of genes can be measured easily after the

introduction of cDNA /oligonucleotide microarrays by using two

essential tools- PCR and hybridization of oligonucleotide to the

complementary sequences, but there are no analogous tools for protein

analysis.

Number of genes identified in in human genome is only about 30,000-35,000.

How can only 35,000 genes encodes for more than 1,00,000 proteins??

Contd…- each gene may encode several proteins by a process K/a alternative

splicing[ one gene makes different m RNA products and hence different proteins]

- A protein may be modified chemically after it is synthesized so that it acquires a different function.

- Proteins interact with each other in complex pathways and network of pathways which may alter their function

All cells express i. genes whose protein products provide essential function

ii. Genes whose protein products provide unique cell specific function.

Thus every organism has one genome and many proteome

Proteome in any cell represents the subset of all possible gene

products.

Interesting facts:

Yeast has more genes than bacteria and fewer than worm & fly.

However fly is much more complicated than worm and has fewer

genes than worms.

Human genome encodes only about twice as many genes as that of

worms.

Thus complexity lies in regulation of genes and functions of protein

products rather than size of genome

Tools of proteomics

separation

Protein mixture protein

digestion digestion

separation

Peptide mixture peptides MS analysis

MS data

database search

algorithms

identification

1.Protein and peptide separations

Done by

One –Dimensional SDS -PAGE

Two –Dimensional SDS-PAGE

Other methods Preparative isoelectric focusing- this technique is analogous to

first step in 2D-SDS-PAGE.

High-performance liquid chromatography- has not become a widely used technique for analytical proteomics.

Protein digestion techniquesProteases that are most widely used in proteomic analysis are

1.Trypsin – by far the most widely used protease in proteomic analysis.

2.Glu-C[ v8-protease]

3.Other proteases and cleavage reagents

Lys-C

chymotrypsin

Asp-N

4.Non specific proteases- subtilysin, pepsin, proteinase K and pronase

Mass spectrometryWhile 2D- gel electrophoresis separates proteins, it doesn’t identify

them. MS is used to identify them which separates charged particles or ions according to mass.

2 types of MS instruments

1. MALDI-TOF – matrix assisted laser desorption ionization-time of flight.

MALDI-is method of ionization

TOF- is a mass analyzer

1. ESI Tandem mass analyzer[ ESI-MS-MS]

ESI- process by which the ions are produced in the source of the instrument

Tandem mass analyzer- are able to perform 2 stage[multistage mass analysis]

To identify individual spots are excised from 2D- gels

subject to ionization to produce population of charged molecules

A mass analyzer then separates the samples molecules based on their mass to charge ratio

a detector will produce a peak for each ion, this peak gives the mass and it represents the amount of ion

computer program will read the complex spectral information from

the mass spectrometry . The program [ database] matches the

information on each peptide’s mass against the mass of theoretical

predicted peptide, based on the known proteins in the database.

• pic

Peptide mass finger printing

is a protein identification technique in which MS is used to

measure the masses of proteolytic peptide fragments. The protein then is identified by matching the measured peptide masses from protein or nucleotide sequence databases.

steps:

-unknown protein is digested with specific protease which cleaves the proteins in a predictable way

- Subject the data base of protein sequence to the same cleavages to generate the virtual peptide mass list to match against .

- Mass of the unknown peptides are measured using MS and matched against the mass list of the database.

peptide mass list

▪

▪

1529.2454

1529.5006

1529.6997

1529.7348 1529.7348

1529.9978 measured mass

1530.2332

1530.4567

▪

▪

▪

Peptide sequence—VGAHAGEYGAEALER

Identification—human Hb alpha

Contd…

This technique has been used to identify unique sets of proteins in the blood, which serve as the markers for different forms of cancer.

As more samples are evaluated , the accuracy will likely to increase because the software will be able to find more accurate peptide patterns correlating to cancer.

Proteomic finger printing holds a great promise as a diagnostic tool for a variety of diseases that produce distinctive pattern of proteins in blood

Interaction proteomics• Proteins are not discrete and independent molecules they need

other proteins or cofactors for their activity.

• Such interactions are necessary for signal transduction, trafficking, cell cycle, and gene regulation.

• Interaction domains and catalytic domains play a role in protein interactions, by binding to a variety of related ligands.

Identifying them is critical in understanding cellular processes.

MS techniques have been developed to study such interactions

Research is being done on what controls which protein interact and for those that interact with multiple complexes how do these interact?

Protein MicroarraysAnother strategy for large scale study of proteins, similar to DNA

microarray.

Procedure :

A very small amount of different purified proteins are placed on a

glass slide in a pattern of rows and columns.

Followed addition of various types of the probe molecules , that

are fluorescent dye labeled ,to the array.

When the probe binds to the label it results in fluorescent signal

that can be read by a laser scanner.

Thus this technique can detect thousands of protein protein

Interactions, can screen the ability of proteins to bind other

proteins in complex, receptors, antibodies, lipids, enzymes,

harmones, specific DNA sequences or small molecules such as new

Drugs.

one of the most promising application of protein micro array is the rapid detection or the diagnosis of the disease by identifying a set of proteins associated with disease

Eg. A microarray containing many different mutant forms of a protein p53.

Researchers screen the immobilized mutant p53 proteins in the microarray for the biological activity , as well as new drugs that can restore its normal tumor suppressing function

Proteomes in different organismsAlthough genome sequence is complete for many organisms we

still do not know what characterizes each of these organism.

eg. Both mouse and human genome contains 30,000 genes. Based on a comparison 99% of genes were conserved in

both species and are thus derived from a common evolutionary

ancestors. The remaining one percent represents genes that

evolved independently in mouse or human.

how can they be so different?

just the presence of genes doesn’t mean the protein is expressed

eg.

Pigs produce a cell surface glycoproteins GAL-proteins that are

present in pig blood vessels. These GAL proteins in are seen as

foreign by human immune system and leads to the rapid

destruction of pig organs when they are transplanted into humans

when human organs was not available.

Humans lack GAL protein, but has gene for making them and they are not expressed in humans.

Another eg.

An experiment in which Proteins from the brains of human and

chimps were compared showed difference in the gene expressed .

And that of the liver and blood showed much less difference.

Eukaryotes have more long proteins and more proteins with

regular secondary structure and less random globular structure and

more loop in proteins when compared to those of other organisms

of different kingdom.

Difference of gene expression not only exists between the different organisms .

Every somatic cell shares the same gene. Differences between tissue types results from differences in the gene expression.

Proteomics and drug discoveryGenome and proteome informations are used to identify the

proteins associated with the disease.

That protein will be used by computer software as a target for

new drug.

- Certain protein implicated in the disease is identified.

- 3D structure of protein in generated using software.

-computer programme design the drugs to interfere with the actions of the protein i.e, a molecule that fits the active site of an enzyme and cannot be released will inactivate proteins.

A good example of this--- identification of the drugs to target and inactivate the HIV-1 Protease. The virus cannot survive without this enzyme. Therefore it is the most effective protein targets for killing of HIV

Applications of proteomics1. mining identification of all of the proteins in a sample

2. Protein expression profiling identification of proteins in a sample as a function of particular state of a cell i.e, two states of a particular systems are compared

3. Protein network mapping to determine protein interaction with each other in a living system

4. Mapping of protein modificationstask to identify how and where proteins are modified post translationally

Proteomics society India

It is formed to provide a forum across

Members - protein scientists from some of the national labs /institutes already engaged in proteomic research

It is inducting the representation from universities , industries and other organisation

Website- psiindia.org

References • Rediscovering biology-protein and proteomics

• Introduction to proteomics-tools for the new biology- DANIEL C.LIEBLER

• Internet

Thank You