What is the main objective of proteomics?
To characterize all of the proteins using a single platform (high-throughput analysis of proteins)
What is the yeast two hybrid used to identify?
Used to identify if two proteins physically interact with each other (detects binary protein interactions)
Describe the steps of carrying out a yeast two hybrid (3 steps)
- Conjugate yeast transcription factor (e.g. Gal4) DNA-binding domain with bait protein (protein of interest). Also conjugate yeast transcription factor activation domain with prey protein.
- Allow Gal4 DNA-binding site with bait protein to bind to promoter site upstream of a reporter gene.
- Introduce Gal4 AD with prey protein to yeast cell.
- The reporter gene only activates if bait and prey proteins physically interact
What is it called a yeast two “hybrid”?
Because bait/DB and prey/AD complexes are hybrids, as they come from different organisms
If a reporter gene is fired up just by the binding of the Gal4 DB/bait protein onto the promoter (i.e. auto-activation), what does this indicate about the bait protein? What do researchers have to account for because of this?
The bait protein is a transcription factor with its own activation domain.
- Researchers need to control for this before the assay to ensure that they don’t get a bunch of false positives.
Yeast two hybrid only identifies…
Proteins that are directly or physically interacting with the protein of interest
What 3 types of proteins would not work well with conventional yeast two hybrid assays?
- If bait protein is a transcription factor
- Membrane proteins don’t work because complex can’t get into the nucleus to turn on a reporter gene
- Kinases don’t work well either because phosphorylation is very fast, so if an interaction between a kinase and substrate is very short then it likely won’t be detected
How are the bait and prey proteins actually brought together in the yeast two hybrid?
Give 2 specific ways that this can be done
By mating a cell with a bait protein/DB plasmid and a cell with a prey protein/AD plasmid and selecting for the presence of both vectors and activation of reporters
Can be done in two ways:
1. Matrix screen: Flood BD strains from test tube onto AD array (systematic/SGA robot), then print the results to indicator plates
2. Library screen: Mix BD strains and library of AD strains from test tubes and select for reporter expression.
Probability that a protein interacts in a yeast two-hybrid assay is a…
Power law
Number of interactions for a protein is related to its…
Essentiality
- In yeast, 93% of proteins have <6 links and 21% of them are essential, while 0.7% of proteins have >15 links and 62% of them are essential
How can we determine the temporal order of protein-protein interactions? (2 ways)
- Party hub
- Date hub
What is a party hub?
Integrate microarray expression data where the assumption is that if all the protein interactors of a hub protein are co-expressed at the mRNA level, then all the interactions to the hub is likely occurring at the same time
- Proteins interacting at same time and space
What is a date hub?
Uncorrelated mRNA expression (seen using microarray) = interactions with hub is occurring at different times
- Proteins interacting at different time and/or space
- Date hubs tend to function in
multiple biological processes and be essential genes
What is a co-immunoprecipitation?
Pull-down one protein and detect presence of the other protein by western blotting (way to confirm that proteins are interacting with each other)
- If both antibodies are detected, then know that the bait and prey are bound together
What is a proteome chip? Describe how these experiments are carried out (4 steps)
A proteome chip is basically a microarray, except on the chip instead of having DNA, you have proteins
1. Express and purify proteins, and spot them onto a nickel-coated microscope slide (with a tag, e.g. GST-HIS6 tag)
2. Detect proteins on array with dyed antibody specific to protein on chip (e.g. α-GST-Cy5), which binds all the proteins on the microarray to prove that they’re there
3. Flood any dye(e.g. Cy3)-labelled molecule onto the array. If Cy3 fluorescence is observed, then flooded protein binds to the specific protein probe.
4. Can then use MEME to see what amino-acid sequence the flooded protein binds to
Design an experiment to identify substrates of kinases (3 steps)
Use a proteome array
1. Create proteome array, and then overexpress/purify the kinase of interest
2. Incubate the proteome array with the kinase and radioactive ATP
3. After incubation, look for radioactive phosphate on the proteome array to identify which proteins on the array were phosphorylated by the kinase
The yeast kineome is an in vitro phosphorylation map of yeast. What was specifically found regarding the specificity of the kinases in this kineome?
Most substrates (73%) were recognized by fewer than three kinases indicating a strong preference of particular kinases for specific substrates
Describe protein microarrays to determine antibody response (3)
- Used for vaccine designing (e.g. for tuberculosis)
- Provides crucial info to find biomarkers for diagnosis, this helps because some people are asymptomatic
- Collect sera from >500 TB patients, isolate antibodies, add IgG secondary antibodies (biotinylated), add streptavidin conjugated fluorescent dye (this is required to see where the antibodies bind), put on microarray, scan
Why would determining antibody/antigen binding not work in a two-hybrid?
You would need a library of billions of antibodies because so many antigens exist
- Not practical for yeast two hybrid because you would have to do it one at a time
What is mass spectrometry? What is affinity purification-mass spectrometry (AP-MS)?
Mass spectrometry: Identify proteins in a complex by measuring mass-to-charge ratios (kg/coulomb) and abundance of ionized proteins/peptides
Affinity purification mass-spectrometry (AP-MS): Identifies proteins directly and indirectly interacting with the protein of interest in a protein complex
What is Tandem Affinity Purification (TAP)? Describe the steps
Systematic pull-down of proteins to isolate complexes and identify the components
1. Bait protein binds TAP tag, which contains calmodulin-binding peptide, a TEV protease cleavage site and protein A
2. Protein A binds to IgG beads in the first affinity column, while prey proteins + contaminants bind to bait protein
3. Cleave TEC protease cleavage site (with protein A and IgG)
4. Calmodulin-binding protein bound to bait/prey proteins binds to calmodulin beads in second affinity column, which allows for eluting of the bait protein/prey protein from contaminants
5. Do mass spec to determine which proteins are interacting with your protein of interest
What are the three basic components of a mass spectrometer?
- Ion source: ionizes peptides or proteins
- Mass analyzer: measures the mass to charge ratio (m/z) of the ionized analytes
- Detector: records the number of ions at each m/z value
