7: neuroproteomics 1 Flashcards
three components of functional genomics
transcriptome = mRNA, proteome = proteins, metabolome = metabolites
what is systems biology?
genomics, transcriptiomics, proteomics, metabolomics then do analysis, databases and mathematical modeling
two types of neuroproteomics
expression: protein expression profiling aka what are all the proteins expressed in a certain condition. function: to look at protein to protein interactions
applications of proteomics (3)
biomarkers discovery, drug development, disease mechanisms
proteomics flow chart (6)
biological sample. prepare for quantitation. protein separation. mass spect analysis. data analysis. protein identification + quantification.
methods for protein separation (2)
2D electrophoresis. 1 and 2 D liquid chromatography
2 dimensions of 2D electrophoresis
first dimension: separating based on pI (isoelectic focusing). 2nd dimension = SDS page separating based on mass
what do you do after separating via 2D electophoresis?
stain the SDS PAGE. then excise spots of interest (differences between the two conditions). trypsin digestion. then proceed to mass spect.
2D-DIGE: stands for? what happens?
differential in-gel electrophoresis: where you run two samples in the same gel, so whatever affects protein migration will affect them in the same way. label with fluorescent dyes to differentiate them.
1D vs. 2D LC
1d: peptide mixture separated based on hydrophobicity. 2D-LC: isoelectric chromatogrphy followed by separation by hydrophobicity = MudPIT aka multidimensional protein identification technology
mass spectrometry: 3 main parts
ion source to make gas-phase ions. mass analyzer to separate ions by m/z. detector to count number of ions for each m/z.
what does MALDI-TOF MS stand for
matrix assisted laser desorption ionization = MALDI. time of flight = TOF
how does MALDI-TOF work
protein sample mixed with acidic matrix. UV laser causes protonation/ionization of peptides. proteins detach from matrix, and enter analyzer.
how does analyzer separate proteins
based on mass/charge ration, but each peptide charged usually with just one positive charge. movement now just based on size. smaller comes out first, bigger ones last.
ESI MS stands for?
electrospray inoization MS
how does ESI MS work
proteins mixed with polar solvent, travel along capillary with charged tip, converted into aerosol. solvent evaporates and you get charged proteins which then enter analyzer
MS/MS stands for?
microsequency by tandem mass spectrometry
what happens in MS/MS : select? fragment?
first mass analyzer selects ions of interest. CID, collision induced dissociation used to fragment selected ions by colliding them with gas (argon)
MS/MS: second mass analyzer? how does fragmentation happen?
measures the fragment ions. occurs along the peptide backbone: each residue successively fragments off in N-C and C-N direction
what are the ion fragments called
“Y” fragments (fragmenting from N terminal). from C terminal would be “b ions”
difference between mass of ion fragments
is equal to the mass of one amino acid: use this to find out sequence
2 techniques to map the neuroproteome?
voxelation and MALDI/MS imaging.
voxelation?
slices of mouse brain cut into voxels (1mm3 cubes) and the extracted proteins subjected to LC-MS analysis.
what happens in MALDI/MS imaging
brain cut into very thin sections. entire section embedded into matrix for ionization, UV bombardment, etc. to see which proteins are in specific sections of the brain
