Exploring proteins and proteomes Flashcards
Proteome
Complete set of proteins expressed. Includes info on how they are modified, how they function, how they interact with other molecules
Which 4 characteristics can be used to separate proteins from each other and other molecules?
- Solubility
- Size
- Charge
- Binding affinity
What is SDS-polyacrylamide gel electrophoresis?
Separates polypeptide chains of proteins under denaturing conditions mainly according to mass.
How can proteins be separated electrophoretically on basis of net charge?
By isoelectric focusing in a pH gradient. Ion exchange chromatography.
How can you separate proteins by size?
Ultracentrifugation and gel-filtration chromatography
What is affinity chromatography?
Uses the high affinity that many proteins have for specific chemical groups. Proteins bind to columns containing beads bearing covalently linked substrates, inhibitors or other specific groups.
How can the mass of a protein be determined?
By sedimentation-equilibrium measurements
How can proteins be detected and quantified?
By highly specific antibodies, particularly monoclonal antibodies, which are especially useful as they are homogeneous.
What techniques are used to detect and quantify proteins with antibodies?
Enzyme-linked immunosorbent assays and western blots of SDS-polyacrylamide gels.
What is immunofluorescence microscopy and immunoelectron microscopy used for?
To localize proteins within cells.
What are matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) used for?
Allow the generation of ions of proteins and peptides in the gas phase. The mass of the ions is then determined.
How is determining the mass of protein and peptide ions useful?
The masses act as protein name tags because mass of protein/peptide is determined by its AA composition and, hence, its sequence.
Why are mass spectrometric techniques, such as tandem mass spectrometry, central to proteomics?
Enables fast and accurate sequencing of peptides. Makes it possible to analyse the constituents of large collections of proteins.
What information does peptide sequence provide?
proteins evolutionary relationships, diseases produced by mutations. Provides clues to conformation and function.
How can polypeptide chains be synthesized?
By automated solid-phase methods. The carboxyl end of the growing chain is linked to an insoluble support. The carboxyl group of the incoming AA is activated by dicyclohexylcarbodiimide and joined to amino group of growing chain.
