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MPharm 2 - PH2114 > Unit 3 - Protein analysis > Flashcards

Unit 3 - Protein analysis Flashcards

1
Q

What are a diverse and rapidly growing segment of the prescription drug market?

A

Protein drugs

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2
Q

Which molecules are central to life and very important in biomedical research?

A

Endogenous proteins

- ones already in the body

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3
Q

How are target proteins harvested following microbial cell lysis?
(Host microbe grows target protein which is then harvested)

A

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

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4
Q

Which chemicals are used to create channels to allow proteins to leak out of a microbe through the cell membranes?

A 
Toluene
Ether
Dimethyl sulphoxide (DMSO)
Methanol
Detergents
Enzymes
Alkali
Chelates
Chaotropic agents
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5
Q

What are the advantages of chemically created channels?

A

they’re Effective

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6
Q

What are the disadvantages of chemically created channels?

A

Introduces additional chemicals into the lysate that can require later (downstream) removal from the purified materials

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7
Q

What is used to break down cell walls of microbes to release proteins?

A

Lysozyme is used for E.coli which has tough cell walls

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8
Q

What are the disadvantages of using enzymatic permeabilisation to release proteins?

A

Slower

Added proteins could cause separation problems downstream

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9
Q

How do mechanical methods release proteins from microbes?

A 
Cell envelope is broken physically
Intracellular components are released into the surrounding medium
- ultrasonification
- freeze-thaw
- liquid shear
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10
Q

What are the advantages of mechanical methods to release proteins from microbes?

A

Less clean up needed downstream

- no addition of reagents

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11
Q

What is lysate?

A

Material obtained after cell lysis

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12
Q

Why does crude lysate need separation, identification and quantitation?

A

Very impure

- especially if extraneous chemicals used to cause lysis

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13
Q

How are proteins different to small molecules in terms of analytical methods?

A

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
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14
Q

Give some examples of protein analysis methods

A 
Fast Protein Liquid Chromatography
- FPLC
Mass Spectrometry
- MS
Electrophoresis
Western Blotting
- IHC
- ICC
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15
Q

What is FPLC?

A

Fast Protein Liquid Chromotography

FPLC is a form of liquid
chromatography used to separate
or purify proteins from complex mixtures

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16
Q

What is Fast protein liquid chromatography (FPLC) Used for?

A

To separate or purify proteins from complex mixtures

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17
Q

What is the difference between HPLC and FPLC?

A

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

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18
Q

What are the three main modes of FPLC?

A 
Ion exchange
- some selectivity based on ionic affinity
Gel filtration
- selective based on molecular size
Immunoaffinity
- highly selective
- based on immuno-recognition
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19
Q

What is ion-exchange chromatography? (a form of FPLC)

A

Separation of ions (proteins) based on charge affinity

Molecules have affinity for counter exchange on stationary phase

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20
Q

How does ion exchange chromatography work?

A

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)

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21
Q

How does anion(positive) exchange chromatography work?

A

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
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22
Q

How does gel filtration chromatography work?( a form of FPLC)

A

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
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23
Q

How is the stationary phase selected in gel chromatography?

A

Based on a knowledge of analyte mass

- available gels with defined upper and lower pore size

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24
Q

Which gels give a higher resolution in gel filtration?

A

Smaller range of pore sizes

- smaller range over which it can separate macromolecules on the basis of their size

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25
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
26
What is immunoaffinity chromatography?
Separation of biochemical mixtures based on highly specific immunoadsorbent interaction - antigen and antibody - enzyme and substrate - receptor and ligand
27
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
28
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 ```
29
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
30
What is MALDI?
Matrix Assisted Laser Desorption/Ionisation
31
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
32
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
33
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
34
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
35
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
36
What are the two main formats of electrophoresis?
Zone electrophoresis | Capillary electrophoresis
37
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
38
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
39
What is analyte migration characterised by?
Electrophoretic mobility
40
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 ```
41
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
42
What is the stationary phase in analyte migration?
The buffer | - needs supporting
43
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 ```
44
What is PAGE?
Polyacrylamide Gel Electrophoresis
45
What is PAGE used for?
A powerful analytical tool in molecular biology to analyse macromolecules - proteins - DNA - RNA
46
How many gels are used in PAGE?
Two - one for stacking - one for resolving molecules
47
How does PAGE work?
In electrical field - large molecules migrate slowly through the gel - small molecules migrate faster - less interaction/hindrance with matrix
48
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
49
What is capillarity?
The ability of a liquid to remain in a narrow channel without the assistance of, and in oppo
50
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
51
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
52
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
53
What is SDS-PAGE?
Sodium dodecyl sulphate Polyacrylamide gel electrophoresis
54
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
55
What is Western Blotting?
Used extensively in molecular biology to detect and quantify proteins in complex mixtures
56
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
57
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
58
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
59
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
60
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
61
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
62
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
63
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

MPharm 2 - PH2114 (32 decks)

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