unit 4.2 Flashcards
Describe tandem mass spectrometry
- MS-1 determines initial mass and separates peptides
- fragmentation of peptide at various points
- MS-2 analyzes mass of breakage products
What is a proteome?
an entire group of proteins produced by a cell/subcellular compartment/tissue/organism
How does a proteome differ from the genome?
it is more complex/larger
includes protein modification and protein complexes
What is a proteome dependent on
- cell type
- developmental stage
- environmental signals
What is the proteomics application of MS
- LC-MS/MS
Describe LC-MS/MS
- Combines liquid chromatography with Tandem MS
- fractionation of Molecules by LC
- Immediate analysis by MS/MS
- Analysis of complex mixture of proteins
What does circular Dichroism spectroscopy do?
- determines the fraction of common secondary structures in proteins.
- measures the differences in absorbance of right vs. left-handed circularly polarized light
- it does not say where proteins are
Molar extinction coefficient
- y-axis measurement for CD spectroscopy
- this coefficient changes for peptide bonds in folded environments
describe x-ray crystallography
- determines the position of atoms in molecules by capturing the scattering of x-rays
- requires alignment of atoms in a crystal
What does the detection of the scattering pattern depend on in x-ray crystallography?
- the atomic arrangement
- resolution down to 1A
What is cryoelectron microscopy?
- 3D microscopy
- gives images of 3D structure of proteins or complexes that fail to crystallize or are too complex for x-ray diffraction analysis
describe how cryoelectron microscopy works.
- a sample of diluted protein solution is applied to a thin layer on a grid and frozen rapidly.
- image is taken by cryoelectron microscope
- images are analyzed by single particle analysis
why is ice crystal formation avoided?
to preserve structure
why are images in cryoelectron microscopy analyzed by single particle analysis?
- b/c proteins are in different orientations
- has a resolution of several angstroms
How does NMR work?
- determines protein structure by analyzing distance and neighborhood of atoms in solution
- requires a high concentration of protein
- used for determining tertiary structure
- utilizes spin of certain isotopes
- absorption spectrum of RF radiation is affected by neighboring atoms (‘chemical shift’)
