15: Proteomics - The global analysis of proteins

Question 1

Proteome

Answer 1

Total set of proteins encoded by a genome or the total protein complement of an organism.

Question 2

Translatome

Answer 2

Total set of proteins that have actually been translated and are present in a cell under any particular set of conditions,

Question 3

Protein characterization

Answer 3

1º structure
- Identification of protein/gene 
  product
- Sequence validation
- Characterization of 
  isoforms/splice variants
- Determination of secondary 
  modifications (PTMs)

2º structure
- Investigation of protein folding
domains
- Disulfide bonds

3º structure
- Determination of 3D structure
- Studies of protein structure
dynamics

4º structure
- Identification of ligand binding 
  sites and kinetics
- Definition of protein-protein 
  interactions

Question 4

2D-PAGE of Proteins

Answer 4

PAGE = Polyacrylamide gel electrophoresis

Polyacrylamide gives smaller gaps in the meshwork than agarose.
=> proteins are smaller than DNA/RNA

Separation by charge (isoelectric focusing) and size (standard SDS-PAGE) in different dimensions.

After isolation:
Protein spots are cut out of gel, digested by protease treatment, and peptides are analyzed by mass spectrometry.

Silver stain is used to detect the proteins.

Question 5

Epitope

Answer 5

Specific 3D portion of a protein that is recognized by an Ab.
Every protein has multiple epitopes.

Question 6

Polyclonal antibodies

Answer 6

Ab’s from an animal that recognize multiple epitopes of a foreign protein

Question 7

Monoclonal antibody

Answer 7

Ab produced by a single clonal B cell from a mouse such that it only recognizes one specific epitope on the protein of interest.

Question 8

Western blot

Answer 8

Detection method in which an Ab is used to identify a specific protein.

Denaturation - heat, SDS, reducing agent
Separated by size - SDS-PAGE or 2D-SDS-PAGE
Transferred to solid membrane by electrophoresis.
Addition of primary and secondary Ab.

Primary Ab binds POI
Secondary Ab binds primary Ab => detection by i.e. fluorescence

Question 9

Chromatography

Answer 9

Can be used to isolate proteins.
Proteins are eluted into multiple fractions.
Proteins absorb light at 280 nm => amount of protein can be measured with UV light.

• Liquid
• Ion exchange
• Hydrophobic interaction columns
• Affinity
• High pressure liquid
  chromatography (HPLC)

Question 10

Mass spectrometry

Answer 10

Technique for measuring the mass of molecular ions derived from volatilized molecules.

Measures mass to charge ratio (m/z) of ions

Question 11

Matrix-assisted laser desorption-ionization (MALDI)

Answer 11

Mass spectrometry where gas-phase ions are generated from a solid sample by a pulsed laser.

• Proteins are crystallized with a
  matrix that absorbs at the
  wavelength of the laser.
• Energy is transferred to proteins
  and ions are released.
• TOF detector measures the time-
  of-flight from the ion source to a
  detector.
  Proportional to the m/z ratio

Post-translational modifications may be detected

Question 12

Electrospray ionization (ESI)

Answer 12

Mass spectrometry where gas-phase ions are generated from ions in solution.

- Liquid sample of protein is held in 
  a capillary tube.
- Exposure to electrostatic field => 
  small droplets are released from 
  the end of the capillary tube. 

• Flow of heated gas within the drift
  zone helps evaporate the solvent
  and release small charged ions.
• Charged grid separates the ions
  by size => impede or help the flow
  of the reactor.

Question 13

Protein-tagging systems

Answer 13

Tagging with another easily recognized molecule allows a researcher to purify or identify the POI without creating a specific Ab for it

Often done genetically
- DNA encoding protein is engineered
to add an extra segment that codes for
the tag.

Question 14

Co-immunoprecipitation

Answer 14

Method of identifying protein-protein interaction by using antibodies to one of the proteins

- Gene for POI is expressed in 
  mammalian cells by transfection.
- POI is isolated from the cytoplasm 
  using Ab's.
- If no Ab is available for the POI => 
  FLAG protein bound to POI and Ab for 
  FLAG tag is used to isolate the protein.
- Beads coated with protein A is added, 
  and bind to Ab's.

Protein complex can be separated via electrophoresis on SDS-PAGE gel.

Question 15

Protein microarray

Answer 15

Microarray of immobilized proteins used for proteome analysis and normally screened by fluorescent or radioactive labelling.

Not to be confused with DNA microarrays to survey DNA-binding proteins, i.e., TFs.

Question 16

ELISA: enzyme-linked immunosorbent assay

Answer 16

Solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured.

Add primary and secondary Ab (labeled). Primary specific for antigen, secondary binds primary.
=> can detect an measure POI