Fractionation Techniques

Question 1

Outline why protein fractionation is a key stem in sample preperation for proteomics analysis and describe fractionation methods that are suitable for (a) biofluids and (b) cells/tissues

Answer 1

• general on fractionation techniques
• proteominer
• immunodepletion
• nanotrap technology
• albuminome
• molecular weight fractionation
• fluorescance activated cell sorting
• laser capture microdissection
• organelle isolation

1. differential centrifugation
2. density-gradient centrifugation
3. differential detergeent fractionation
4. free-flow electrophoresis
5. immunoaffinity purification

Question 2

1. Proteominer

Answer 2

• decreases the dynamic range of proteins within a sample, dilutes abundant proteins and concentrates and enriches low abundance proteins
• based on a combinatorial ligand library 10^9 - 10^12 unique peptide ligands, 1 unique hexapeptide ligand per bead
• highly specific affininty interactions between proteins and ligands ensure binding the greatest number and variety of proteins
• limited bead capacity allows maximum concentration of rare species while dilution of high abundant species
• can be used for differential expression analysis and is compatible with current downstream protein analysis techniques

Question 3

2. Immunodepletion

Answer 3

• for removing a target molecule from a mixture by adding an antibdoy targeting the molecule of interet
• simple 2 buffer system that only utilises the centrifuge but yields low collection volume
  DISADVANTAGES
• sample may be diluted during elution
• efficiancy will vary dramatically from antibody to antibody
  ever-deeper mining of the proteome requires an ever-expanding set of immunodepletion products

Question 4

3. Nanotrap Technology

Answer 4

• ceres nanotrap is a carbon-based capture-particle that can be as small as 100 nm, comprising a molecular sieve portion and an analyte binding portion that acts as bait for low abundance analytes
• analytes bonding to bait and concentrate in nanotrap whilst larger molecules blocked out by sieving properties, decreasing the amount of high-abundance proteins present in samples
• concentrate and preserve highly labile analytes, prevents degradation of scarce and short-lived biomarkers during sample processing
• enriches and concentrates low abundance proteins in complex biofluid samples like proteominer
• does not utilise antibodies for immunodepletion or immunoprecipitation like proteominer
• simultaneously harvest multiple low-abundance proteins form a single sample
• compatible with protein analysis techniques e.g western blotting, mass spec analysis

Question 5

4. Albuminome

Answer 5

• collection of proteins that bind to albumin in serum
• HSA (human serum albumin) most abundant human protein, represents over 50% of total protein circulating in bloodstream
• peptides and proteins bound to serum albumine and molecule itself yield important information for disease diagnosis and management
• atleast 35 different proteins are carried by albumin along with drugs circulating in the bloodstream

Question 6

1. molecular weight fractionation

Answer 6

• uses spin columns with specific molecular cutoff membranes

Question 7

2. fluorescance activated cell sorting (FACS)

Answer 7

• allows us to pull out a pure population of cells from a mixture of cell types
• cells are placed into a flask and forced through a small nozzle and travel down it producing drops at fixed distances from the nozzle. as the cells flow down the stream of liquid they are scanned by a laser. some of the laser’s light is scattered which is used to count the cells and measure the size of the cells.
• seperating a subpopulation of cells using this technique can be done by tagging those of interest with an antibody linked to a fluorescent dye.
• electrical charge is also used to sort the drops into 3 seperate sample tubes resulting in 3 pure subpopulations of cells sorted by charge

Question 8

3. laser capture microdissection

Answer 8

• uses a low-energy laser beam and special transfer film to lift a desired cell out of the tissue section leaving all unwanted cells behind
• laser cuts around boundaries of e.g tumour
• heat from laser activates reson on transfer film and binds to cut out tissue section that is loosened from rest of tissue/cellular population
• when it solidifes, will be connected to transfer film
• will only work well with a good pathologist that can identify where tumour cells reside

Question 9

4. organelle isolation

Answer 9

TRADITIONAL
1. differential centrifugation
- operates cia sequential centrifugation of the cell or tissue homogenate
- based on differences in size and density
- fractions prone to contamination with organelles with similar sedimentation velocities

2. density-gradient centrifugation
   - seperates organelles based on continuos and discontinuous gradients
   - use of various media with different osmolarities, viscosities or densities
   - after cell homogenate added to top of medium and centrifuged, organele focuses in the gradient where its density equals the density of the surrounding medium (isopycnic point)
   - 2 types; continuous gradient and discontinuous gradient
3. differential detergent fractination
   - use of buffers of increasing stringency, often different detergent-containing buffers
   - seperation of proteins in native state according to 4 compartments (cytosolic, membrane and membrane organelle-localised, soulble and DNA-associated nuclear, cyoskeletal proteins)

RECENTLY DEVELOPED
4. free-flow electrophoresis
- organelle seperation based on their net global isoelectric charge or electrophoretic mobility
- purififed organelles retain their intactness and functionality

5. immunoaffinity purification
   - isolation of membrane proteins with magnetic beads
   - very fast, simple protocols but is very expenisve

Question 10

1. general on fractionation techniques