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Proteome: ALL proteins actually expressed in a single cell, tissue, organ, organism at a particular time and under particular conditions
Proteome: ALL proteins actually expressed in a single cell, tissue, organ, organism at a particular time and under particular conditions
Protein abundance: NOT predictable from mRNA levels (translation rates and protein degradation are not taken into account).
Posttranslational modifications and proteolytic cleavage are not detectable by measuring mRNA levels.
Proteins usually work in complexes and their localization in a cell is highly regulated, although not detectable by mRNA levels.
Proteome comprises: all proteins actually expressed in a single cell, tissue, organ, organism at a particular time and under particular conditions, so it is important to understand everything that contributes to this such as:
Exosome: protein complex Containing Exoribonucleases with 3’- RNA degradation activity and RNA binding proteins.
Exosome degrades RNA trimmings : the leftovers of RNA processing, incorrectly processed RNAs, RNAs with defects, Only high-quality RNAs Translated
regulation of translation initiation: due to translational control of previously existing mRNAs.
modifications of the translational machinery: affect specific translational efficiency.
Proteins that bind to specific sequences in mRNAs and thereby changing translation efficiency
protein processing
Proteolytic cleavage: can activate proteins or protect cells from deleterious effect of the mature protein.
posttranslational modifications (phosphorylation, glycosylation),
-(PTMs): adding of chemical molecules to a protein
Glycosylation: covalent attachment of carbohydrate chains (of 12 or more residues) to oxygen (in Golgi) or nitrogen (in ER) of amino acids
Glycosylation effects: Proper folding of proteins, target proteins to particular sites in cells, Cell to cell recognition and interaction, increase protein stability
Phosphorylation: Reversible PTM, at OH groups of ser, thr and tyr side chains, conformational change that can either activate or inactivate Protein function.
-Phosphorylation regulates: cell cycle, growth, apoptosis and signal transduction pathways.
protein interaction: Regulation of proteins requires interaction (molecular machines contain many proteins)
-Interaction leads to function leads to Organisation leads to species Proteome differences
protein degradation : Polyubiquitin marks protein for degradation,
Polyubiquited Proteins: bind to the proteasome
Exosome: protein complex with binding sites in the cap for polyubiquitinated proteins and internal proteases that degraded target proteins
Ubiquitin : 76 AA protein attached to Lysine residues
Target proteins: denatured proteins and proteins with specific sequences in the target protein N-terminus (determine life span of the protein).
Proteomics: Identify proteins, function, interaction, modifications, localisation within cells.
Protein profiling: separate individual proteins from each other, Identify proteins, usually using mass spectrometry.
Top down: directly examine individual proteins using MS.
Bottom up: break up proteins into peptides by sequence-specific protease treatment and examine peptides by MS.
2D PAGE: two dimensional polyacrylamide gel electrophoresis
2D PAGE process: 1st dimension seperate by charge, 2nd dimension seperate by MW, image analysis, excise spots of interest, MS
- Condition a and condition b Isolate and solubilise proteins from cells
- Isoelectric focussing on narrow or Separate by charge, broad range pI strips 1 st dimension
- Soak in SDS Transfer onto SDS gel
- SDS-PAGE 2nd dimension separate by size/MW Staining options: 1. Silver 2. Coomassie 3.Fluorescent 4. Autoradiography
- Condition A Condition B Image analysis
- Excise spots of interest
- Mass Spectrometric Identification of spots
➔ identification of differences by comparison by eye is difficult • Solution: use computer pattern recognition software, use DIGE
2D DIGE: Label proteomes with cy3 and cy5, Pool samples with internal standard cy2, run in same gel, 3 markers spectrally different, the fluorescence of markers different allowing distinction between 2 treatments, cy3 and cy5 fluorescence values determined for each spot in relation to cy2, differentially expressed spots excised and run through MS.
-Labelling of proteomes with Cy3 or CY5 fluorescent markers. (equal MW)
-A pooled sample of both proteomes with a third fluorescent marker CY2, this serves as an internal standard.
All samples are mixed and run in the same 2D gel. ➔ no running or gel differences.
-three markers are spectrally different, allowing distinction between the two treatments:
▪ Cy3 and Cy5 fluorescence values are determined for each spot in relation to the fluorescence of
the standard mix labelled with Cy2.
- Differentially expressed spots excised from gel and analyzed by MS
HPLC: Separation of analytes by distributing those between a mobile and a stationary phase, using a property of functional group to interact and separate analytes.
Hydrophobic interaction chromatography separates by hydrophobicity: stationary matrix has hydrophobic groups that interact with the hydrophobic regions of molecules.
Normal/reversed-phase chromatography: separates by polarity. The stationary phase contains either highly polar (normal phase) or highly non-polar (reverse phase) functional groups that interact with molecules according to their polarity level.
Ion-exchange chromatography: separates molecules by net electric charges. The stationary phase carries either positive or negative functional groups and retains molecules of the opposite charge.
Affinity chromatography: separates molecules by binding interactions. Resins carry small molecule with which proteins can interact.
Size-exclusion chromatography: separates molecules by their size.
Mass spectrometry of protein peptides: identifies compounds such as peptides by mass to charge (m/z) ratios
MS: 1st ionization, separation of compounds with different m/z ratios, detection of individual compounds, identification using database entries or standards.Requires
▪ first ionisation of the compound
▪ followed by separation of compounds with different m/z ratios
▪ then detection of the individual compounds
▪ and finally identification using database entries or standards
Proteins digestion: Typically by trypsin, cleaves on the Cside of Arg and Lys i.e. generates peptides having R or K at the C-terminus
- Isotope-coded affinity tag (ICAT) a tag that contains:
• at one end a chemical groups that can be attached to a peptide.
• linker regions made up of hydrocarbon chains containgin the 12C or 13C isotope.
• a final group, that allows separation of labelled from unlabelled proteins by affinity chromatography.
o One protein set is labelled with the 12C containing tag, other set with 13C isotope tag.
o Mixed then MS
o Proteins in the two sets due to the different carbon, have slightly different m/z ratios ➔ protein pairs from the two set will run closely together but can be distinguished by their slightly different m/z ratios.
o Peak height comparison allows the relative abundance of proteins in the two samples to be determined
ionisation of peptides,
Electron spray ionisation: Peptide dissolved in polar volatile liquid and pumped through stainless steel capillary, Strong voltage applied at capillary tip along with flow of nebulizing gas, forming charged droplets, droplet solvent evaporates in vacuum assisted by a drying gas (desolvation) , droplets undergo coulomb fission, desolvation and coulomb repeated until only peptides/proteins left carrying charge.
- Nebulize (aerosolize) forming charged droplets
desolvation: droplet solvent evaporates in vacuum assisted by a drying gas
coulomb fission and desolvation: repeated until there are only peptides/proteins left carrying the charge.
Coulomb fission= Breaking up because electrostatic repulsion of like charges in decreasing droplet size > surface tension holding the droplet together in the decreasing droplet
MALDI: Matrix assisted Laser desorption Ionisation
-Peptides generated by peptidase digest --> co-crystallised in UV absorbing matrix
MALDI PROCESS: Matrix absorbs energy from a short UV laser pulse --> Concentrated heat produces localised explosion of sample/matrix --> evaporate forming gas phase --> vapor contains peptide ions --> proton transfer in resulting plasma results in peptide cation formation.
Quadrupole Mass analyser: Four parallel metal rods, opposing rod pair is connected together electrically
radio frequency (RF) voltage with a DC offset voltage is applied between one pair of rods and the other pair à electromagnetic field allowing only ions with a specific m/z ratio - resonant ion - to go to the detector
Non-resonant ions: collides with rod
➔ Many different fields are applied in very fast succession per second
➔ Mass analyser scans a mass range
Time of flight mass analyser: Ions accelerated in an e- field by transferring KE to the ions
TOF dependant on mass of ions: EK = 1/2MV^2 , with EK constant all ions have the EK or 1/2MV^2 so velocity is dependant on its mass, Ions with higher mass have lower velocity = smaller time of flight
-check gene sequence is correct, check exon intron boundaries were correctly predicted/located leads to identifying alternative splicing events
Antibody arrays = analytical protein array
Antibodies immobilised on array. Each antibody specific for a single protein.
▪Proteome sample is applied to the array. Proteins with an antibody on the array will bind, others are washed away.
▪ Amount of binding depends on abundance of a protein in the proteome ➔ quantitation is possible
▪ Captured proteins are detected using fluorescently labelled, secondary antibodies that binds to all the proteins in the proteome ➔ fluorescence intensity can be used to quantify amount of bound protein
o Protein interaction:
Bimolecular fluorescence complementation
- uses two halves of a fluorescent proteins genes, translationally fuse each half to a different protein being tested for interaction à interaction of 2 tested proteins à reconstitute a functional fluorescent protein.
-Excitation with a laser causes light energy to be absorbed by fluorescent protein à emits light of a diff wavelength detectable by fluorescence microscope. (no interaction no emmision)
FRET: resonance energy transfer
-Uses two different fluorescent protein, the 2 genes encoding for these fluorescent proteins are translationally fused to the two proteins tested for interaction.
-Interaction of proteins brings the two fluorescent proteins close together, Laser light is absorbed by one of the fluorescent probes à protein then emits a light of a different wavelength à absorbed by the second fluorescent protein à emits light of a diff wavelength that can be observed under a fluorescent microscope .
Co-IP: Co-immunoprecipitation
-Cell expresses a bait protein fused to a GFP tag in cells à fusion protein interacts with another protein à extract proteins à add anit-GFP antibody (AB) coupled to protein a agarose beads à antibody binds to GFP protein à precipitate contains AB bound to GFP fused bait protein and interacting proteins
phage display: Uses a vector based on a bacteriophage genome, vector contains a gene that encodes for a phage coat protein.
(Genes encoding the test proteins are inserted into the phage coat protein gene à library of test protein genes fused to coat protein gene.)
vector is transformed into E.coli causing production of the phage, Phages display the test proteins on the outside of the coat.
-POIs are immobilized within wells of a microtiter plateà Phages displaying test proteins with a POI will bind to the POI non-binding phages are washed away.
protein interaction arrays,
Print bait proteins onto a array – > add a mix of fluorescently labelled test proteins -> wash away non-bound proteins à visualize and analyse
yeast-2-hybrid system
- Gal4: transcriptional activator of GAL gene,DNA binding domain (DBD) and gene activation domain (AD)
▪ DBD binds to an upstream activator sequence (UAS) in gene promoters
▪ AD is responsible for activating transcription by interacting with RNA pol
▪ Separate DBA(Bait)and AD (Prey) domain
Fuse these coding regions to either of the proteins à if the proteins interact transcription of a selectable marker is activated.
o The bait vector has the GAL4 DNA binding domain (DB)
o The prey vector has the GAL4 transcription activation domain (AD
False positive due to auto-activation: some proteins can bind the DNA and if these are fused to the AD they will induce transcription
Protein interaction maps: show all of the identified interactions of a set of proteins
Nodes: a protein
Edges: interacting proteins
Hubs: Proteins that form many interactions
-Party Hubs: interact with all partners simultaneously. Removal has little effect on network structure
- Date hubs: interact with different partners at different times. Removal breaks network into a series of small networks
Disease modules: specific genes when defective, give rise to the same disease often encode for proteins that are part of disease modules in a interaction network
Comorbidity: Overlaps between different disease modules