Unit 3 - Protein analysis Flashcards
What are a diverse and rapidly growing segment of the prescription drug market?
Protein drugs
Which molecules are central to life and very important in biomedical research?
Endogenous proteins
- ones already in the body
How are target proteins harvested following microbial cell lysis?
(Host microbe grows target protein which is then harvested)
Chemically created channels
allowing proteins to leak
out through the cell membranes.
- Toluene, ether, dimethyl sulphoxide (DMSO), methanol, also used, detergents, enzymes, alkali, chelates and chaotropic agents
- Effective, but introduce additional chemicals into the lysate that can require later (downstream) removal from the purified materials.
Enzymatic permeabilisation
- Enzymatic permeabilization with agents such as lysozyme is used for E. coli, which has tough cell walls. Generally slower, and these added proteins could cause separation problems downstream.
Mechanical methods
- Mechanical methods The cell envelope is broken physically, and the intracellular components are released into the surrounding medium.
- Ultrasonication, freeze–thaw and liquid shear
Preferred as less clean-up is needed downstream, as no addition of reagents
Which chemicals are used to create channels to allow proteins to leak out of a microbe through the cell membranes?
Toluene Ether Dimethyl sulphoxide (DMSO) Methanol Detergents Enzymes Alkali Chelates Chaotropic agents
What are the advantages of chemically created channels?
they’re Effective
What are the disadvantages of chemically created channels?
Introduces additional chemicals into the lysate that can require later (downstream) removal from the purified materials
What is used to break down cell walls of microbes to release proteins?
Lysozyme is used for E.coli which has tough cell walls
What are the disadvantages of using enzymatic permeabilisation to release proteins?
Slower
Added proteins could cause separation problems downstream
How do mechanical methods release proteins from microbes?
Cell envelope is broken physically Intracellular components are released into the surrounding medium - ultrasonification - freeze-thaw - liquid shear
What are the advantages of mechanical methods to release proteins from microbes?
Less clean up needed downstream
- no addition of reagents
What is lysate?
Material obtained after cell lysis
Why does crude lysate need separation, identification and quantitation?
Very impure
- especially if extraneous chemicals used to cause lysis
How are proteins different to small molecules in terms of analytical methods?
Size
- generally macromoles
Chemical nature/functionality
Proteins adopt secondary/tertiary configuration
Lability (quality of being likely to change)
- many proteins are susceptible subtle changes or degradation
- can affect or negate pharmacological action
- whichever lysis, separation and analytical method is used
- target protein must remain unaltered
Give some examples of protein analysis methods
Fast Protein Liquid Chromatography - FPLC Mass Spectrometry - MS Electrophoresis Western Blotting - IHC - ICC
What is FPLC?
Fast Protein Liquid Chromotography
FPLC is a form of liquid
chromatography used to separate
or purify proteins from complex mixtures
What is Fast protein liquid chromatography (FPLC) Used for?
To separate or purify proteins from complex mixtures
What is the difference between HPLC and FPLC?
Particle sizes are larger in FPLC
- beads
Selective stationary phases
Low pressure
Glass or plastic columns
- The main interest is in the nature of the stationary phases used to achieve purification
What are the three main modes of FPLC?
Ion exchange - some selectivity based on ionic affinity Gel filtration - selective based on molecular size Immunoaffinity - highly selective - based on immuno-recognition
What is ion-exchange chromatography? (a form of FPLC)
Separation of ions (proteins) based on charge affinity
Molecules have affinity for counter exchange on stationary phase
How does ion exchange chromatography work?
Molecules have affinity for countercharge on stationary phase (ion exchanger)
- cationic exchange resin is negative (e.g COO-, SO3-) and
exchanges positively charged ions (cations)
anionic exchange resin is positive
and exchanges negatively charged ions (anions)
How does anion(positive) exchange chromatography work?
Protein mixture added to top of column
-ve charged protein retained as its attracted to stationary phase.
+ve charged/neutral protein eluted as its not attracted
Mobile phase changed - stronger eluting power - buffer pH -ve charged protein eluted because there's no affinity therefore proteins have been separated
How does gel filtration chromatography work?( a form of FPLC)
Molecules are separated based on size
Glass column filled gel which acts as the stationary phase.
The gel is made up of porous beads
- channels of predetermined size
Molecules larger than pore size cannot enter the pores and elute together as the first peak in the chromatogram
- total exclusion
Molecules that can enter the pores will have an average residence time in the particles that depends on the molecule’s size and shape
Different molecules therefore have different total transit times through the column
- selective permeation region
Molecules smaller than the pore size can enter all the pores - longest residence time on the column as they stay longer within the pores and elute as the last peak in the chromatogram
How is the stationary phase selected in gel chromatography?
Based on a knowledge of analyte mass
- available gels with defined upper and lower pore size
Which gels give a higher resolution in gel filtration?
Smaller range of pore sizes
- smaller range over which it can separate macromolecules on the basis of their size
Which gels give a lower resolution in gel filtration?
Wider range of pore sizes
- will permit fractionation of a larger range of sizes as an initial step when the approximate molecular mass is unknown
What is immunoaffinity chromatography?
Separation of biochemical mixtures based on highly specific immunoadsorbent interaction
- antigen and antibody
- enzyme and substrate
- receptor and ligand
How does immunoaffinity chromatography work?
Stationary phase with immobilised antigen
Protein mixture/lysate added
Elution
- immunoaffinity reaction with correct recognition
- others pass through
Bound protein released and collected
Give examples of Mass Spectroscopy methods to analyse proteins?
ESI - followed by quadrupole MALDI - followed by TOF ELECTROSPRAY IONISATION - produces ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it is comparatively mild – low fragmentation
What is Electrospray ionisation (ESI)?
Produces ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol
- especially useful in produces ions in macromolecules
- comparatively mild
- low fragmentation
- readily linked to HPLC-LCMS
What is MALDI?
Matrix Assisted Laser Desorption/Ionisation
How does MALDI work?
Sample dispersed in matrix
- 3,5-dimethoxy-4-hydroxycinnamic acid
Sample irradiated by N2 laser
Matrix absorbs some radiation, protecting the biomolecule from being destroyed, facilitating vaporisation and ionisation
Allows the MS analysis of labile biomolecules that would degrade using other ionisation methods
- biopolymers
- proteins
- peptides
- sugars
- large organic molecules
- polymers
- dendrimers
- other macromolecules
What is TOF?
Time Of Flight
TOF analysis is based on accelerating a group of ions towards a detector having imparted the same amount of energy
How does TOF work?
Because the ions have the same energy, yet a different mass, the ions reach the detector at different times
- smaller ions reach the detector first
- greater velocity
- larger ions take longer
- lower velocity
What factors affect the arrival time of an ion at the detector in MALDI-TOF?
Mass
Charge
Kinetic energy of the ion
As the chromatogram develops
- first compounds to appear have the smallest mass
- the last compounds have the largest mass
What is electrophoresis?
Separation of molecules by differential migration in an applied electrical current
- mostly applicable to charged/dipolar analytes
- single phase
- an electrolyte/buffer solution
- stationary
What are the two main formats of electrophoresis?
Zone electrophoresis
Capillary electrophoresis
How does zone electrophoresis work?
Electrolyte retained by an inert porous supporting medium
- usually sheet form
- NOT THE STATIONARY PHASE
Application of current results in components of a mixture migrating and being separated into individual bands
- cations migrate towards the cathode
- anions migrate towards the anode
What is analyte migration?
Analytes migrate in response to applied current
Analyte ceases to move when attractive force balanced by friction or isoelectric focussing
- migration in the electric field until isoelectric point reached
- no overall charge
What is analyte migration characterised by?
Electrophoretic mobility
What factors affect electrophoretic mobility?
Analyte - charge - pKa - size - functionality Electrolyte - ionic strength - temperature - ampholytes can change direction of migration due to pH change Electrolyte buffered to - precise pH - ionic strength - temperature
What is electroosmosis?
Migration of neutral compounds or even -ve compounds to -ve terminal
Analytes dissolved in aqueous medium
- solvated by multiple water moleculs
Analyte are transported because the water molecules responding the the current
What is the stationary phase in analyte migration?
The buffer
- needs supporting
What supporting media are used to support the buffer in analyte migration?
Paper/Cellulose acetate - cheap but fibrous - ionic groups - tailing - poor resolution Agar and agarose - extracted from seaweed - linear polysaccharide composed for agarobiose - hydrogel forms a gel-like semi-solid matrix Polyacrylamide - extensively used in PAGE - synthetic polymer - control of cross linking - tailored pore size
What is PAGE?
Polyacrylamide Gel Electrophoresis
What is PAGE used for?
A powerful analytical tool in molecular biology to analyse macromolecules
- proteins
- DNA
- RNA
How many gels are used in PAGE?
Two
- one for stacking
- one for resolving molecules
How does PAGE work?
In electrical field
- large molecules migrate slowly through the gel
- small molecules migrate faster
- less interaction/hindrance with matrix
How does DNA analysis work?
DNA fragmented using restriction enzymes
Fluorescent intercalating agent added
- wedges into grooves of DNA
Sample loaded at cathode
- negative
Fragments migrate in electric field towards anode
Separation according to fragment size
- smaller fragments migrating faster
Under fluorescence view ethidinium bromide indirectly with the DNA fragment
Bands compared with other samples to establish a match or otherwise
What is capillarity?
The ability of a liquid to remain in a narrow channel without the assistance of, and in oppo
How does capillary electrophoresis work?
Capillary electrophoresis is different to gel electrophoresis as capillaries allow application of a much stronger electric field.
This allows faster separation.
Fragments separated by size with the smallest fragments moving fastest.
Uses capillarity to retain electrolyte the stationary phase, in an inverted U-shape column, to bridge the two reservoirs
Sample added to negative reservoir
Strong electroosmosis overwhelms electrostatic repulsion
What are the limitations of PAGE?
Band is present in a small 3 dimension volume of gel
- gel has width and height, but also depth
We cannot probe much more than qualitatively
How can the band from PAGE be analysed?
Transferred all molecules within the band out of the gel onto another material where it remains on the surface
What is SDS-PAGE?
Sodium dodecyl sulphate Polyacrylamide gel electrophoresis
What is sodium dodecyl sulphate?
An ionic surfactant that is added to polyacrylamide gels, buffers or sample prior to analysis
- analyte proteins becomes covered in the negatively charged SDS and therefore always move towards the positively electrode with current applied
- allows sample to be placed at negative end
- maintains polypeptides in a denatured state
- allowing separation of proteins in a linear arrangement by their molecular weight
What is Western Blotting?
Used extensively in molecular biology to detect and quantify proteins in complex mixtures
What happens to proteins after they are separated by SDSPAGE?
Electrophoresis is used to transfer (blot) them onto the surface of a membrane
- nitrocellulose
- PVDF
- polyvinylidene fluoride
How is Western Blotting set up?
The gel and membrane are sandwiched between blotting papers soaked in buffer and the set is compressed between two parallel electrodes in a cassette
Current is passed at right angles to the gel
- proteins migrate to the membrane surfaces
- retaining their separation
How does Western Blotting work?
SDS PAGE
Blocking
- prevents interactions between the membrane and the antibody used for detection
Blotting
- protein bands transferred from the porous gel onto non-porous nitrocellulose or PVFD membrane
- the membrane is placed on top of the gel and a stack of tissue papers placed on top of that
- the proteins move from within the gel onto the membrane while maintaining the organisation they had within the gel
- as a result of this “blotting” process the proteins are exposed on a thin surface layer for detection
Probing
- the membrane is probed for the protein of interest with either by
- modified primary antibody which is linked to a reporter enzyme which when exposed to an appropriate substrate drives a colorimetric reaction and produces a colour
- primary antibody followed by a secondary antibody linked to reporter enzyme
Visualisation
- after the unbound probes are washed away, the western blot is ready for detection of the probes that are labelled and bound to the protein of interest
- colourimetric
- substrate reacts with the reporter enzyme stains the nitrocellulose membrane a specific colour
- chemiluminescence
- substrate reacts that luminescent when exposed to the reporter on the secondary antibody
Image is analysed/quantitated by densitometry
What do the results of Western blotting show?
The higher the analyte concentration the more the bound AgAb so the more AgAb conjugate hence the darker the signal intensity
Why are loading controls needed for Western blotting?
In order to draw any conclusions from a Western blot one has to be sure that the observed differences are only due to altered protein expression levels rather than gel loading or protein transfer errors
What are the loading controls that are used in Western blotting?
Must be present across all cell types or tissue types that are used in the experiment and should not change due to the experimental procedure
What is Beta-actin?
Expressed within all eukaryotic cell types and not affected by cellular treatments and run in tandem
Protein of interest normalised against the beta-actin band
How is electro spray ionisation different to MALDI TOF
ESI is different from other ionization processes (e.g. matrix-assisted laser desorption/ionization (MALDI)) since it may produce multiple-charged ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments
