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?• 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?• 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 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 Proteomics Genomics mRNA Protein Fractionation Primary Protein products 2-D ElectrophoresisFunctional protein products Proteomics Protein Post-Translational Identification Modification Determination of gene
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 Methodologies• Analysis of protein expression patterns• Analysis of protein Sequence Information• Analysis of protein structure/function relationships
Proteomic 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)
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
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 are1. Preparation of the sample2. Solubilization3. Reduction4. IPG-IEF5. Equilibration6. 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
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.
• 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
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).