Block A Lecture 4 - Proteomics Flashcards
What is proteomics?
Research focussed on the analysis of large sets of proteins, such as how they interact with each other within a cell
(Slide 7)
What 2 methods does proteomics rely on?
Biochemical methods of affinity tagging and mass spectrometry
(Slide 7)
What has proteomics resulted in the creation of?
Organised databases of protein interaction maps
(Slide 7)
What is a protein interaction map?
A graphical representation of the physical or functional interactions between proteins in a biological system.
(Slide 9)
What do protein interaction maps help researchers understand?
How proteins work together in various pathways
(Slide 9)
What is Skp1?
It stands for S-phase kinase-associated protein and it is part of the SCF ubiquitin ligase complex, which functions to catalyse ubiquitination
(Slide 10)
What other functions does Skp1 have outside of ubiquitination?
Functions at the origin of cell replication
Functions in cell cycle progression
Functions in methionine synthesis
Functions in kinetochore
(Slide 10)
Protein interaction maps can be extremely complicated and complex, making it hard to understand the complexities of the cell or interactions. How do scientists get around this?
They tend to now consider smaller sub-sections of the protein interactions maps which are focussed around a few proteins of interest
(Slide 11)
What is a protein interaction network?
A network which maps protein interactions. Proteins are usually represented by a dot or box in a 2-dimensional network, with a straight line connecting proteins which have been found to bind to each other
(Slide 12)
What are the steps of proteomics?
- Sample preparation
- Sample separation
- Spot picking and polypeptide band selection
- Trypsin Digestion - generation of peptide fragments
- Mass spectrometry
- Data analysis and protein identification
- Post-analysis validation
- Exploration of applications in health and disease
(Slide 13)
How are whole cell extracts prepared for proteomics?
Via standard lysis with Laemmli buffer
(Slide 16)
What are 3 methods which are used to prepare protein complexes for proteomics?
They are precipitated by one of the below methods:
Immuno-precipitation (using an antibody specific to protein of interest)
Affinity-based capture
Use of overexpressed bait proteins (such as GFP, GST or HA tagged baits)
(Slide 16)
What do methods used to prepare protein complexes for proteomics generate?
A sub-set of proteins which specifically interact with the protein of interest
(Slide 16)
What are 3 chemicals which can be used in “labelling by staining”?
Coomassie brilliant Blue R-250
Colloidal; Coomassie Blue G-250
Silver (Silver ions)
(Slide 17)
Why is silver hazardous to use in “labelling by staining”?
As it is not compatible with mass spectrometry
(Slide 17)
Compare 1D and 2D electrophoresis.
1D electrophoresis usually only separates proteins by a single property, usually mass where as 2D separates proteins by 2 properties, usually mass and charge
Both use SDS-page though 2D electrophoresis also needs isoelectric focussing first
(Slides 19 - 22)
What is isoelectric focussing?
A method of separating proteins according to their isoelectric points in a pH gradient, with the isoelectric point being the pH at which a protein carries no net charge (aka the pH at which a protein becomes immobile in an electric field)
(Slides 20 - 22)
What is polypeptide band selection?
The process of choosing specific protein bands from a gel after electrophoresis to undergo further analysis, with selected bands being proteins of interest picked based on their size, abundance or role in a biological process
(Slide 23)
How are bands of interest removed during polypeptide band selection after 1D and 2D electrophoresis?
1D electrophoresis - bands of interest are removed with a scalpel
2D electrophoresis - a spot picker machine which automatically picks selected protein spots from stained or detained gels is used
(Slide 23)
What is trypsin digestion?
An enzymatic process used in proteomics which uses trypsin to break down proteins into smaller peptides by digesting them in the gel.
(Slide 24)
Why does the size of peptides produced by trypsin digestion vary?
As the presence of trypsin digestion sites is amino acid specific
(Slide 24)
What is mass spectrometry?
A method which enables the precise measurement of the molecular weight of many substances. It can be used to identify peptide fragments generated by trypsin digestion
(Slide 25)
What limitation of mass spectrometry lead to the development of “soft” ionisation techniques of mass spectrometry?
As the studied substance needs to be intact in the gas phase of mass spectrometry for protein analysis
(Slide 25)
What are 2 examples of “soft” ionisation techniques of mass spectrometry?
Matrix assisted laser detection of desorption / ionisation (MALDI)
Electrospray ionisation (ESI)
(Slide 25)
