15 - Proteomics Flashcards
Proteome
the total set of proteins encoded by a genome or the total protein complement of an organism
one problem in proteomics is the individuality of different proteins. one purification method does not work for all proteins.
translatome
The total set of proteins that have actually been translated and are present in a cell under any particular set of conditions
The disparity between the transcriptome and proteome can be summarized in 7 bullet points
These modifications may all vary depending on the conditions and activities of other genes/proteins.
1) some RNA molecules are non-coding and do not give rise to any protein products
2) some promary RNA transcripts undergo alternative splicing; therefore the same gene may give rise to multiple protein products
3) levels of mRNA may not correlate with protein levels due to differential rates of mRNA translation or degredation
4) the activity of many proteins is regulated after stranslation by addition or removal of acetyl, phosphate, AMP, SDP-ribose, or other groups
5) the activity of many roteins is altered after translation by chemical modification of amino acid residues
6) many proteins are processed after translation: for example, by proteolytic cleavage or addition of sugar or lipid residues to give glycoproteins or lipoproteins.
7) proteins themselves may be degraded and vary greatly in stability
Isolating and quantifying proteins
the procedure depends on the protein, but in general the first step is to break open the cells. How this is done depends on the organism. Could use detergents, or glass beads/blenders. freeze + thaw.
DNases and RNases are usually added after the opeing of the cell to digest the nucleic acids.
How to determine the total amount of protein in a sample?
many methods. 2 widely used coorimetric assays rely in copper chemistry and a protein-binding dye, respectively.
biuret regant = a solution containing KOH, hydrated copper (II) sulfate, and sodium potassium tartrate. The proteins in a solution will react with these component to produce a blue/violet complex that is quantified by measuring absorbance at 540 nm, thus determining the protein concentration.
The Lowry assay:
adds Folin phenol reagent to enhance the color and sensitivity and measures absorbance at 650-750 nm)
The other method (Bradford assay) uses Coomassie Blue. Protein smaple is mixed with the reagents. As coomassie blue reacts with positively charged AAs in proteins >=3kD, the color develops.
Gel electrophoresis of proteins
nucleic acids = neg charges -> move towards potistive pole.
Proteins are not so easy :/ POs, neg and neut AA exists, leading to multiple possible charges of the proteins.
As some proteins will move towards the positive, some toward the negative, and some stay (neutral), the protein samples are usually loaded in the middle of a gel = native protein electrophoresis. Sometimes used for purifying proteins without deactivating them.
To separate by size, the proteins are boiled with SDS (sodium dodecul sulfate). This destroys the folded 3D structure of the protein (=denatured).
SDS has a hydrophobic tail with negative charge at the end. Tail wraps around protein, neg charge dangles in water. the protein is unrolled and covered sith SDS-molecules, giving it a negative charge. number of neg charges is proportinal with the length of the protein.
Small sulfhydryl reagents can be added to disturb the disulfide bonds (tertiary structure).
gel is of polyacrylamide as the proteins are smaller than DNA fragments. the PAGE-gel is stained with coomassie blue or silver compounds.
2D-PAGE of proteins
2D: separate by charge in the first dimention and size in the other.
Isoelectric focusing is used in the first dimension and separates native proteins based on their original charge. A pH gradient is set up along a cyndrical gel, and proteins wander until they reach their isoelectric point (neutral charge).
Standard PAGE for size separation
After separation, the protein sports are cut from the gel, digestes with protease treatment and the resulting peptides are analyzes by mass spectrometry.
the presence or absence of a protein is detected by a sensitive silver stain. QUantity: Coomassie Blue or fluorescent dyes. The gel is then scanned with a laser. Many dyes are possible.
Antibodies are essential proteomics tools
epitope = specific 3D portion of a protein that is recognized by an anibody. Every protein has multiple epitopes.
POlyclonal Ab can be made by immnizing an animal for the protein, then harvesting the Ab by collecting blood serum from that animal. NOT suited for proteomic experiments in general, as they always have some contamination.
Monoclonal antibodies can be made the same way, and Ab-secreting B cells are removed and fused to tunor cells (myeloma) (= hybridoma) which can live indefinetly in vitro.
Western Blotting of Proteins
Detection method in which Ab is used to identify a specific protein
1) denature sample with heat, SDS and a reducing agent
2) Separate by size with SDS PGAGE or 2D-SDS-PAGE.
3) the proteins are electrophoretically transferred to a solid membrane such as nitrocellulose. Electrophoresis moved the proteins from the gel onto the nitrocellulose where the proteins adhere.
To detect a protein, an Ab for that protein must be availible. The nitrocellulose membrane has many nonspecific sites that can bind ABs, so before adding the primary Ab these sites must be blocked by a nonspecific protein solution (such as rehydrated powdered milk, BSA). The primary Ab is then added to the membrane where it only binds the POI. A secondary Ab with a detection system is used to detect the primary Ab/protein.
Isolating proteins with chromatography
liquid chromatigraphy: technique that separates a mizture of proteins through a column containing different solid metals.
High pressure liwuid chromatography uses high pressure to propel the sample through the column.
ion exchange chromatography: technique that separates a mizture of protein based upon their native charge. The resin is either pos or neg charged, (an- or cation exchange). The ions on the resin binds to oppositely charged proteins in the sample mixture and hold them to the column. Any proteins with the same or neutral charge pass through the column without binding. The attached proteins can later be removed by changes in pH.
Hydrophobic interaction columns have resins that bind hydrophobid proteins. they may be removed by adjusting the salt concentration.
Reverse-phase chromatography is a related method since the resin also bins hydrophobic proteins, but the interactions between the resin and hydrophobic proteins is much stronger. The proteins are eluted with organic solvents rather than salts.
affinity chromatography uses resins with a ligand specific for the POI. Thus, only the POI binds to the column and the rest pass through.
For all chromatography methods, the bound proteins are eluted in multiple fractions. Proteins absorb 280 nm UV, and the amount of protein in each fraction can be monitored with UV light.
Mass spectrometry for Protein Identification
MS measures the m/z ration (mass to charge) of ions and allows derivation of the molecular weigth.
MALDI = matrix-assisted laser desproption ionization. gas-phase ions are generated from a solid smaple by a pulsed laser.
ESI = electrospray ionization = gas-phase ions are generated from ions in solution.
MALDI
1) sample protein/peptide is crystallized along with a martix that absorbs the wavelength of the laser. Matrix materials are usually aromatic acids.
2) the laser excites the matrix material, which transfers the energy to the crystallized protein
3) the energy then releases ions, the size and charge of which are unique to each protein.
4) the ions are accelerated by a high-voltage electric field and travel in a vacuum through a tube to the detector.
5) The TOF (time of flight) detector measures the time for an ion to fly from the ion source to the detector.
TOF is proportional to the sqrt(m/z).
ESI
refers to the generation of gas-phase ions from ions in solution. a narrow capillary tube allows droplets of liqueid to emerge onto a strong electrostatic field. The solvent evaporates and the droplet breaks up. Repeated evaporation and splitting of the droplets eventually releases separate ions (with single or multiple charges) that are accelerated towards a mass analyzer by an electric field. Mass analyzers such as quadrupole or ion-trap detectors are normally used with ESI mass spectrometers.
An advantage to ESI is that it can be directly coupled to liquid separation techniques such as capillary electrophoresis or HPLC. Also, a parent ion can be isolated and fragmented into daugther ions, so allowing more detailed analysis of molecules. This is known as tandem mass spectrometry (MS/MS). It allows two parent ions with the same mass to be distinguished
Protein tagging systems
usually done genetically (the DNA encoding the proteins is engineered to ass an extra segment that codes for the tag.) The gene must therefore be cloned and carried on a suitable vector.
The hybrid gene is often expressed in E. coli or a cultured mammalian cell, and the protein is purified by a method that binds to and isolated the tag seq. The tag portion can be removed after purification, or a protein array can be constructed by attaching the tagged protein to a chip via the tag
First common tag = His tag (polyhistidine). 6 tandem histidine residues. can be added to N- or C-terminal end. Binds very tightly to nickel ions: His-tagged proteins are therefore purified using a column with Ni(2+).
FLAG = short peptide (DYKDDDDK) that is bound to a specific Ab.
Anti-FLAG Ab is available and can be attached to a suitable resin for use in column purification.
“Strep” tag = 10 AA peptide that mimics the 3D structure of biotin. It is bound by avidin or straptavidin
Full-length proteins used as fusion tags (497)
the tag protein coding seq is normally places 5’ to the GOI in order to ensure good translation initiation. Three of the most populat are Protein A (staphylococcus) and maltose-binding protein (E. coli). These generally give fusion protein products that are stable, soluble and behave well during purificaiton.
The columns for purification specifically binds the tag proteins.
polylinker = a stretch of artificially synthetized DNA that contains cut sites for seven or eight widely used REs
