BY MUHAMMED RASHID AKM.pharm pharmaceutics

What is a Proteome?What is a Proteome?

• The terms proteomics and proteome were coined by Wilkins et al. in 1994 to describe the entire collection of proteins encoded by genomes in the human organism.

• Proteomics differs from protein chemistry at this point since it focuses on multiprotein systems rather than individual proteins and uses partial sequence analysis with the aid of databases.

Why is the Proteome Important?Why is the Proteome Important?

• It is the proteins within the cell that:– Provide structure– Produce energy– Allow communication– Allow movement– Allow reproduction

• Proteins provide the structural and functional framework of cellular life

What is Proteomics?What is Proteomics?

• Proteomics refers to the systematic analysis of protein profiles of entire cells, tissues, organisms, or species.

• It represents the protein counterpart to the analysis of gene function.

• Proteomics is an attempt to describe or explain biological state and qualitative and quantitative changes of protein content of cells and extracellular biological materials under different conditions to further understand biological processes.

Aims of Proteomics • Detect the different proteins expressed by

tissue, cell culture, or organism using various techniques.

• Store those information in a database

• Compare expression profiles between a healthy cell vs. a diseased cell

• The data comparison can then be used for testing and rational drug design.

Proteomics vs GenomicsProteomics vs Genomics

• DNA sequence does not predict if the protein is in an active form

• RNA quantitation does not always reflect corresponding protein levels

• Multiple proteins can be obtained from each gene (alternative splicing)

• Genomics cannot predict post-translational modifications and the effects thereof

• DNA/RNA analysis cannot predict the amount of a gene product made (if and when)

Proteomics and genomics are inter-dependent

Genome Sequence

mRNA

Primary Protein products

Functional protein products

Determination of gene

Genomics

Proteomics

Proteomics

Protein Fractionation

2-D Electrophoresis

ProteinIdentification

Post-TranslationalModification

Why is Proteomics Important?Why is Proteomics Important?

• Identification of proteins in normal and disease conditions

– Investigating epidemiology and taxonomy of pathogens

– Analysis of drug resistance

• Identification of pathogenic mechanisms

– Reveals gene regulation events involved in disease progression

• Promise in novel drug discovery via analysis of clinically relevant molecular events

• Contributes to understanding of gene function

Proteomic MethodologiesProteomic Methodologies

• Analysis of protein expression patterns

• Analysis of protein Sequence Information

• Analysis of protein structure/function relationships

Proteomic MethodologiesProteomic Methodologies

• Complex protein mixtures are separated by 2-D gel electrophoresis

• Then individual proteins are isolated from spots and are identified by using mass spectrometry

• Individual proteins are sequenced, followed by database searches

• Bioinformatics

Schematic representation of basic proteomics analysis

Protein mixture

separation

proteins

digestion

peptides

MS analysis

MS dataSoftware assisted database search

peptide sequence identification

Protein separation

• First step in proteomic research

• The biological sample for analysis is first pulverized; homogenized, sonicated, or disrupted to form a mixture containing cells and sub cellular components in a buffer system

Proteins are extracted from this mixture using

• Detergents: SDS, CHAPS

help membrane proteins to dissolve and separate from lipids.

• Reductants: DTT, thiourea

reduce disulfide bonds or prevent oxidation

• Denaturing agents: urea, acids

alter the ionic strength, pH of the solution and destroy protein–protein interactions, disrupting secondary and tertiary structures

• Enzymes: DNAse, RNAse , Digestion is achieved by enzymes. Protease inhibitors

are often used to prevent proteolytic degradation.

Extracted proteins are separated by following techniques

• 1D-SDS-PAGE (1-dimensional sodium dodecyl sulfate-polyacrylamide gel

electrophoresis)

• 2D-SDS-PAGE (2-dimensional sodium dodecyl sulfate-polyacrylamide gel

• electrophoresis)

• IEF (isoelectric focusing)

• HPLC (high performance liquid chromatography)

• Size exclusion chromatography

• Ion exchange chromatography

• Affinity chromatography

Gel Electrophoresis

• Motion of charged molecules in an electric field.• Polyacrylamide gel provides a porous matrix

– (PAGE – Polyacrylamide Gel Electrophoresis)

• Sample is stained with comassie blue to make it visible in the gel.

• Sample placed in wells on the gel

1-D Gel Electrophoresis• Separation in only 1 dimension: size.

• Smaller molecules travel further through the gel then large molecules, thus separation.

Steps

• 1. Preparation of a loading buffer containing a thiol reductant (e.g., DTT)d SDS

• 2. Dissolving protein in the loading buffer

• 3. Binding of SDS to protein to form a protein-SDS complex

• 4. Applying to the gel

• 5. Applying high electric voltage to the ends of the gel

• 6. Migration of the protein-SDS complex

• 7. Formation of bands on the gel in order of molecular weight

2DE• most effective way of separating proteins

• Help to identify diseases-specific proteins, drug targets, indicators of drug efficacy and toxicity.

• separation of post-translationally modified protein from the parent one is usually achieved by 2DE.

• Several thousands of different proteins can be separated from each other in one gel.

• 2-DE separation is conducted based on

the electrical charge and molecular weight (size) of the proteins

• First step is to separate based on charge or isoelectric point, called isoelectric focusing.

• Then separate based on size (SDS-PAGE).

steps 2-DE are

1. Preparation of the sample

2. Solubilization

3. Reduction

4. IPG-IEF

5. Equilibration

6. SDS-PAGE

Preparation of the Samples for 2-DE

• The method with minimum modification should be chosen, otherwise artifactual

spots may form on the gel and mislead the operator

• Serum, plasma, urine, cerebrospinal fluid (CSF), and aqueous extracts of cells and tissues are often require no pretreatment.

• They can be directly analyzed by 2-DE following a solubilization step with a suitable buffer (e.g., mostly phosphate buffered saline, or PBS).

• Liquid samples with low protein concentrations or large amounts of salt should be desalted and concentrated prior to 2-DE.

• Desalination can be achieved by dialysis or liquid chromatography

Solubilization

• In order to avoid misleading spots on the 2-DE profile and to remove salts, lipids,polysaccharides, or nucleic acids interfering with separation,

• solubilization procedure involves disruption of all noncovalently bond protein complexes into a solution of polypeptides

Reduction of Proteins• involves reduction of disulfide bonds in the

protein samples.

• DTT or β-mercaptoethanol are the most widely used reducing agents.

• noncharged reducing agents (e.g., tributyl phosphine: TBP*) have been preferred recently.

ISOELECTRIC FOCUSING (IEF)

• The isoelectric point is the pH at which the net charge of the protein molecule is neutral

• Proteins in mixture are separated based on their isoelectric points (pI) following the voltage application.

• With the commercially available IEF apparatus, proteins can be separated into 12–20 fractions.

• Equilibration step:

• . gels are often equilibrated prior to

second dimension analysis in order to allow separated proteins to interact with SDS.

• This interaction will provide migration during SDS-PAGE analysis.

• Equilibration can be achieved by incubating the strips for 15 minutes in 50 mM Tris buffer of pH 8.8 in the presence of SDS, DTT,urea, and glycerol

SDS-PAGE

• Second Dimension.

• Separation by size.

• Run perpendicular to Isoelectric focusing.

• The only unresolved proteins after the first and second dimensions are those proteins with the same size and same charge – rare!

• 2D-PAGE Analysis

• Gel matching, or “registration”, is the process of aligning two images to compensate for warp.

List of 2-D GEL DATABASES

• One can find an extensive list of such databases by following these links.

• We would discuss a few “Interesting ones”.

• World 2-D PAGE

• NCIFCRF

• DEAMBULUM-Protein Databases

• Ludwig Institute of Cancer Research

• Phoretix

• LINKS

• Z3 system (Compugen) - http://www.2dgels.com/

• Melanie3 (SIB) - http://us.expasy.org/melanie/

• ProteomWeaver (Definiens) - http://www.proteomweaver.com/

• PDQuest (Bio-Rad) - http://www.biorad.com/

• Delta2d (Decodon) - http://www.decodon.com/

PROTEIN DIGESTION reasons behind this approach

• MS instruments used for the analysis of separated proteins run for peptides with fewer errors.

• Because the greater the mass of the protein, the greater the possibility of obtaining inaccurate results.

• difficult to perform MS on very large and hydrophobic proteins.

• Sensitivity of mass measurements of peptides is superior to that of proteins.

• 5. Currently available MS instruments are much more suitable for peptide

• analysis and the data obtained can be directly used for comparison with

• protein sequences derived from proteome databases.

• enzymes used for digestion.

• Proteases are the most widely used

enzymes.

Trypsin is the most frequently used serine protease

• Glu-C, so-called V8-proteases, is an endoprotease digesting proteins at carboxyl side of glutamate residues in the buffer solutions

• Nonspecific proteases such as subtilysin, pepsin, proteinase K, or pronase are also used in proteomics

• Cyanogenbromide (CNBr) is the most widely used chemical digestion

agent. It cleaves proteins at methionine residues

Identification of separated protein• Second step in proteomic study.

• The basic identification process is analysis of the sequence or mass of six amino acids unique in the proteome of an organism, then to match it in a database.

MASS SPECTROMETRY (MS) FOR PROTEOMICS

• The ion producing source

• A mass analyzer: converts components of a mixture into ions based on their mass/charge ratio (m/z ratio)

• A detector to detect the resolved ions.

• most frequently used instruments of MS-based proteome analysis are

MALDI-TOFF

matrix-assisted laser desorption ionization time of flight.

• MALDI refers to the source of ionization whereas TOF indicates type of the mass

analyzer.ESI Tandem Ms

• electrospray ionization mass spectrometry performed in multistage

• based on the production of multiply charged

ions from proteins and peptides.

Matrix-assisted laser desorption/ionization (MALDI)

Electrospray ionization (ESI)

• The resultant ion is propelled into a mass analyzer by charge repulsion in an electric field.

• Ions are then resolved according to their m/z ratio.

• Information is collected by a detector and transferred to a computer for analysis

TECHNIQUES USED FOR STRUCTURAL PROTEOMICS

• aims the determination of three-dimensional protein structures in order to better understand the relationship between protein sequence, structure,and function

• NMR and x-ray crystallography are used t determine the structure of macromolecule

• To obtain optimal results, protein should possess minimum 95% purity.

• .

• the molecule under investigation should be purified by gel or column separation,

dialysis, differential centrifugation, salting out, or HPLC prior to structural analysis.

X-ray crystallography• X-ray crystallography is used to determine

the tertiary structure of a protein

• Much information about flexibility of protein structure has also come from x-ray

• crystallography data. the production of crystals for x-ray studies can sometimes cause structural anomalies. They might mask native architectural features.

• membrane proteins are not readily

amenable to existing crystallization methods

NMR• NMR measures proteins in their native state• Precise crystallization, which is often

difficult, is not necessary for conducting structural analysis by NMR

• NMR is increasingly being recognized as a valuable tool, not only in three-dimensional structure determination, but also for the screening process

• Proteins with large molecular weight (up to 30 kDa) can be analyzed

The most significant advantages of NMR spectroscopy are

• it reveals details about specific sites of protein molecules without a need to solve the whole structure.

• It is sensitive to motions of most chemical events which in turn provides direct and indirect examination of motions within micro-time scale (milliseconds to nanoseconds, respectively).

Differential Protieomics