Proteomics Protein-Protein Interaction

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Proteomics & Protein-Protein Interaction

Proteomics:

Proteomics Proteomics is the large-scale study of protein, particularly their structures and functions or The study of the full set of proteins encoded by a genome . This term was coined to make an analogy with genomics (identified the sequence of DNA in species ) and proteomics (systematic study of the complete complement of proteins of organisms). The genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment.

Why Proteomics? :

Why Proteomics? Whole Genome Sequence –complete, but does not show how proteins function or biological processes occur. Post-translational modification –proteins sometimes chemically modified or regulated after synthesis. Proteins fold into specific 3-D structures which determine function. Gain insight into alternative splicing. Aids in genome annotation.

The Virtue of the Proteome :

The Virtue of the Proteome • Proteome = protein compliment of the genome •Proteomics = study of the proteome •Protein world = study of less abundant proteins • Transcriptomics = often insufficient to study functional aspects of genomics

Roles of Proteomics :

Roles of Proteomics • Proteins are the instruments through which the genetic potential of an organism are expressed = active biological agents in cells • Proteins are involved in almost all cellular processes and fulfill many functions • Some functions of Proteins –enzyme catalysis, transport, mechanical support, organelle constituents, storage reserves, metabolic control, protection mechanisms, toxins, and osmotic pressure

Types of Proteomics :

Types of Proteomics 1. Expression Proteomics 2. Structural Proteomics 3 . Functional Proteomics

1. Expression Proteomics :- :

1. Expression Proteomics :- This is the qualitative and quantitative study of the expression of total proteins under two different conditions . For example, expression proteomics of normal cells and diseased cells can be compared to understand the protein that is responsible for the diseased state or the protein that is expressed due to disease. Using this method disease-specific protein can be identified and characterized by comparing the protein-expression profile of the entire proteome or of the sub proteome between the two samples

2. Structural Proteomics :- :

2. Structural Proteomics :- Structural proteomics is related to the structural aspects of protein including the three-dimensional shape and structural complexities, of functional proteins . This includes the structural prediction of a protein when its amino acid sequence is determined directly by sequencing or from the gene with a method called homology modeling . It can map out 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 ribosomes , membranes, or other cellular organelles and to characterize or predict all the proteins and protein interactions that can be possible between these proteins and protein complexes . Structural proteomics of a specific organelle or protein complex can give information regarding supra-molecular assemblies and their molecular architecture in cells, organelles, and in molecular complexes.

3. Functional Proteomics:- :

3. Functional Proteomics:- This is related to macromolecular networks involved in the life activities of a cell. It will be possible to identify specific protein molecules and their role in individual metabolic activities and their contribution to the metabolic network that operates in the system. This forms one of the major objectives of functional proteomics. For example, the recent elucidation of the protein network involved in the functioning of a nuclear pore complex has led to the identification of novel proteins involved in the translocation of macromolecules between the cytoplasm and nucleus through these complex pores.

Different Approaches for Proteome Purification and Protein Separation for Identification by MS:

Different Approaches for Proteome Purification and Protein Separation for Identification by MS A. Separation of individual proteinsby 2-DE B. Separation of protein complexesby non-denaturing 2-DE C. Purification of protein complexesby affinity chromatography + SDS-PAGE D. Multidimensional chromatography . E. Fractionate by Organic Solvent–separate complex protein mix, hydrophobic membrane proteins.

Techniques of Proteomics :- :

Techniques of Proteomics :- 1. Proteinse parathion techniques: - Protein extraction - Cell fractionation and protein separation by electrophoresis - Chromatography protein purification 2. Protein detection techniques:- -Two-dimensional electrophoresis ( isoelectric focusing electrophoresis, capillary electrophoresis etc) 3. Image analysis for proteomics experiments. 4. Protein and peptide identification by peptide fingerprinting, amino acid sequence analysis, database search. 5. Characterization of protein post-translational modification. 6. Functional proteomics.

2-Dimensional Protein Electrophoresis (2-DE) :

2-Dimensional Protein Electrophoresis (2-DE) Purify Proteins from desired organelle, cell, or tissue Separate Protein mixture in 1-D by pI Separate Protein Mixture in 2-D by MW Stain Gel, Data Analysis Protein Identification by MS

Plant Protein Extraction and Fractionation:

Plant Protein Extraction and Fractionation

Basic Components of a Mass Spectrometer:

Basic Components of a Mass Spectrometer Intel Ion Source Mass Analyzer Detector Instrument Control System Vacuum System Data Systm

Types of Mass Spectrometers:

Types of Mass Spectrometers • MALDI-TOF • ESI TANDEM MASS SPEC INSTRUMENTS 1. Quadropole Mass Analyzers 2. Ion Trap Mass Analyzers 3. TOF Mass Analyzers

Proteomics Applications :- :

Proteomics Applications :- The molecular functions of a protein can be inferred from either its sequence or structure information. Sequence-based function inference methods annotate molecular function of a protein from its sequence homologues. Most genome-wide functional annotations are carried by using sequence alignment tools such as BLAST, or motif/profile-based search tools (e.g. PROSITE, PFAM, etc.). Protein domain patterns are assuming high importance in the analysis of the macromolecular functionality mainly when correlated with the relative gene function. Typically these kinds of studies are implemented by confronting a particular set of input sequences with databases of profiles derived from the analysis of specific set of proteins.

Protein-Protein Interactions :

Protein-Protein Interactions It is physical contacts with molecular docking between proteins that occur in a cell or in a living organism. Protein –Protein interaction are include most of biological processes like gene expression, cell growth, proliferation, nutrient uptake, morphology, motility, intercellular communication and apoptosis. Types of protein-protein interactions :- 1. Stable Interaction 2. Transient

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1. Stable interaction:- Stable interactions those which are purified as multi-subunit complexes, and the subunits of these complexes can be identical or different. Hemoglobin and core RNA polymerase are examples of multi-subunit interactions that form stable complexes. 2.Transient interactions :- These are expected to control the majority of cellular processes. As the name implies, transient interactions are temporary in nature and typically require a set of conditions that promote the interaction, such as phosphorylation , conformational changes or localization to discrete areas of the cell. Transient interactions can be strong or weak, and fast or slow. While in contact with their binding partners, transiently interacting proteins are involved in a wide range of cellular processes, including protein modification, transport, folding, signaling, and cell cycling.

Biological effects of protein-protein interactions:- :

Biological effects of protein-protein interactions:- The result of two or more proteins that interact with a specific functional objective can be demonstrated in several different ways. Alter the kinetic properties of enzymes, which may be the result of subtle changes in substrate binding or allosteric effects. Allow for substrate channeling by moving a substrate between domains or subunits, resulting ultimately in an intended end product Create a new binding site, typically for small effectors molecules Inactivate or destroy a protein Change the specificity of a protein for its substrate through the interaction with different binding partners; e.g., demonstrate a new function that neither protein can exhibit alone Serve a regulatory role in either an upstream or a downstream event

Methods to Analyze Protein-Protein Interactions :

Methods to Analyze Protein-Protein Interactions Method Protein-Protein Interactions Co- Immunoprecipitation (co-IP) Stable Pull-Down Assay Stable Crosslinking Protein Interaction Analysis Transient Label Transfer Protein Interaction Analysis Transient Far-Western Blot Analysis Moderately stable

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