Sept 18 Purification, Detection and Characterisation of Proteins 1

Question 1

what are some examples of physical and chemical properties?

Answer 1

mass or size (and shape)
density
electrical charge
binding affinity

Question 2

what are some separation methods?

Answer 2

centrifugation
electrophoresis
chromatography

Question 3

what is centrifugation and how does it work?

Answer 3

centrifugation is the rapid spinning of the centrifuge tube, which generates a centrifugal force that is measured in units of Earth’s Gravity (1g)
this force acts on particles (all the way down to molecular size) suspended within the liquid medium of the centrifuge tube (the supernatant, usually aqueous)
if the particles are denser than the suspending medium, the g force will push the particles to the bottom of the tube
if the particles are less dense, they will float toward the top of the tube
if the particles have the same density, they will stay where they are
usually (but not always) the particles we are interested in are denser than the suspending medium
pellet is formed when the particles move towards the bottom
based on mass/size

Question 4

what does the rate at which the supernatant is cleared of particles depend on?

Answer 4

it depends on the size/mass of the particle (for particles of similar shape)
the size unit calculated this way is called the Svedburg, or S
(example: 40S ribosomes)

Question 5

what is electrophoresis and how does it work?

Answer 5

depends on the charge:mass ratio
in a free solution at a given electric field, the direction of migration is determined by the net charge, and the speed of migration is determined by the net charge/mass ratio
in gel electrophoresis the migration of molecules may be impeded by the gel (larger molecules are impeded more than small molecules)

Question 6

what does SDS do? (the anionic detergent sodium docecyl sulfate)

Answer 6

developed in the 1960s and still used in molecular biology labs
SDS denatures proteins by the interaction of its hydrophobic tail with hydrophobic amino acid side chains, disrupting the oil drop structure of proteins
hydrophobic tail of SDS binds not only to hydrophobic residues, but also to itself, so it coats the polypeptide chain in a uniform layer of SDS molecules
all negatively charged, all repel each other, further disrupts and unfolds protein
completely denatures individual polypeptides but also separates the chains of multimeric proteins into individual denatured polypeptides

Question 7

what is the isoelectric point?

Answer 7

the pH at which the sum of all charges is 0

Question 8

what does the isoelectric point depend on?

Answer 8

depends on the amino acid composition of each protein

Question 9

how is the pH gradient established in isoelectric focusing?

Answer 9

pH gradient is established using special buffers (ampholytes) immobilized in acrylamide gel
proteins subjected to electrical field migrate

Question 10

how are proteins separated using isoelectric focusing?

Answer 10

the same proteins displays different charges in different regions of the strip
proteins accumulate at the isoelectric point
basic proteins: negatively charged, migrate towards the anode
acidic proteins: positively charged, migrate towards the cathode

Question 11

how does mass spectrometry work?

Answer 11

an analytical, not preparative method (because samples are destroyed and cannot be recovered)
high precision determination of the charge-to-mass ratio of ionized molecules
1. produce dispersed (individual molecules) ions in a gas phase
2. measure the acceleration of the ions in an electric or magnetic field
3. acceleration depends on the mass/charge ration (m/z)

each amino acid has a characteristic molecular weight (MW)
if the molecule carries a single charge, then m/z=MW

Question 12

what is a commonly used process for generating gas-phase ionized molecules?

Answer 12

electrospray ionization

Question 13

what is proteomics?

Answer 13

the analysis of biological protein samples by mass spectroscopy and bioinformatics (computer analysis of DNA and protein sequences) in order to identify the population of proteins present in any given subcellular organelle

Question 14

what is differential centrifugation and how does it work?

Answer 14

separation of water-soluble proteins from insoluble cellular material
a starting mixture is poured into a tube and spun at a rotor speed
cell organelles such as nuclei as well as large unbroken cells or large cell fragments collect as a pellet at the bottom
the soluble proteins remain in the liquid above the pellet (the supernatant)
supernatant is poured off, and then it or the pellet can be subject to other purification and analytical techniques

Question 15

explain how rate-zonal centrifugation works

Answer 15

1. on the basis of differences in mass, water soluble proteins can be separated by centrifugation through a solution of increasing density, called a density gradient
2. concentrated sucrose solution is commonly used to form a density gradient in a centrifuge tube (higher concentration of sucrose and higher solution density towards the bottom)
3. protein mixture is placed on top of a sucrose density gradient tube and subjected to centrifugation
4. each protein in the mixture migrates down the tube at a rate controlled by the protein’s physical properties
5. all proteins start from the thin layer of the sample that was placed at the top of the tube and separate into bands of proteins of different masses as they travel at different rates through the gradient
6. centrifuged just long enough to separate molecules of interest into discrete bands, also called zones

Question 16

what is sedimentation rate influenced by?

Answer 16

strongly influenced by particle mass, but variations in shape also affect sedimentation rates

Question 17

how does charge affect the movement in electrophoresis?

Answer 17

a molecule with a greater net charge will move faster towards an electrode of the opposite polarity

Question 18

how does shape of molecule affect rate of migration through the gel in electrophoresis?

Answer 18

long, asymmetric molecules migrate more slowly than spherical ones of the same mass

Question 19

what is polyacrylamide gel electrophoresis (PAGE)?

Answer 19

electrophoretic separation of proteins performed in polyacrylamide gels
cross linked chains of acrylamide monomers that form polyacrylamide chains
the pore size can be adjusted by adjusting the concentration of polyacrylamide

Question 20

how can SDS and PAGE be combined?

Answer 20

the amount of SDS that binds to the protein is proportional to the length of the polypeptide chain and independent of the sequence
as SDS bound proteins move through the polyacrylamide gel, they are separated according to size by the sieving action of the gel
chain length, proportional to mass, is the principal determinant of the migration rate of proteins
all the SDS bound protein are negatively charged (artificial charge distribution), so they all migrate towards same pole but different speed

Question 21

how can the molecular weight of a protein be estimated with gel?

Answer 21

comparing the distance that it migrates through a gel with the distance that proteins of known molecular weight migrate in the same gel

Question 22

how do time of flight analysers work in mass spectroscopy?

Answer 22

TOF instruments exploit the fact that the time it takes an ion to pass through the length of the mass analyser before reaching the detector is proportional to the square root of m/z (smaller ions move faster than larger ones with the same charge)

Question 23

how do ion traps work in mass spectroscopy?

Answer 23

tunable electric fields are used to capture ions with a specific m/z and to sequentially pass the trapped ions out of the mass analyzer onto the detector in order of their m/z rations

Question 24

how can the sequence be deducted from tandem or MS/MS instruments

Answer 24

any given parent ion in the original mass spectrum can be chosen for further analysis
chosen ions are transferred into a second chamber in which they are broken into smaller fragment ions by collision with an inert gas, and then the m/z and relative abundances of the resulting fragment ions are measured in a second MS analyser
permits the sequence of short peptides to be determined, because fragmentation occurs primarily at peptide bonds

Question 25

how can the effect of SDS be sped up?

Answer 25

by heating the proteins with SDS
As the proteins melt, the SDS binds and keeps them
from reforming

Question 26

how does 2-dimensional electrophoresis work?

Answer 26

1st dimension: separated based on isoelectric point
2nd dimension: separated based on weight

A combination of isoelectric focusing and SDS
polyacrylamide gel electrophoresis
It is
isoelectric focusing in one dimension followed by
SDS polyacrylamide gel in a second dimension.
polyacrylamide gel that was
an isoelectric focusing gel and we run it up as a
little tube where it has a low pH at one end and a
high pH at the other end.
proteins all separate according to isoelectric point
We take the gel and incubate
it for a brief period of time in SDS. Thus, the SDS penetrates into the gel, denatures
and binds to all the proteins.
This gives the proteins similar charge to mass ratio and
makes them negatively charged since they are bound to the SDS.
We take that SDS
soaked isoelectric focusing gel and turn it sideways and put it on top of a slab of an
SDS polyacrylamide gel. We layer that isoelectric focusing gel on top and then we
turn on the electric field where we have a negative at the top end of this gel and
positive at the bottom. The SDS protein complexes are negatively charged and they
migrate into SDS polyacrylamide gel. The proteins will migrate according to their
molecular weight. In other words, the smaller proteins will run fast, and the larger
proteins will run slow. Finally, the reason why this technique is so powerful in protein
analysis is that this allows a great separation between different polypeptides,
allowing us to visualize hundreds of protein spots simultaneously.