7: neuroproteomics 1

Question 1

three components of functional genomics

Answer 1

transcriptome = mRNA, proteome = proteins, metabolome = metabolites

Question 2

what is systems biology?

Answer 2

genomics, transcriptiomics, proteomics, metabolomics then do analysis, databases and mathematical modeling

Question 3

two types of neuroproteomics

Answer 3

expression: protein expression profiling aka what are all the proteins expressed in a certain condition. function: to look at protein to protein interactions

Question 4

applications of proteomics (3)

Answer 4

biomarkers discovery, drug development, disease mechanisms

Question 5

proteomics flow chart (6)

Answer 5

biological sample. prepare for quantitation. protein separation. mass spect analysis. data analysis. protein identification + quantification.

Question 6

methods for protein separation (2)

Answer 6

2D electrophoresis. 1 and 2 D liquid chromatography

Question 7

2 dimensions of 2D electrophoresis

Answer 7

first dimension: separating based on pI (isoelectic focusing). 2nd dimension = SDS page separating based on mass

Question 8

what do you do after separating via 2D electophoresis?

Answer 8

stain the SDS PAGE. then excise spots of interest (differences between the two conditions). trypsin digestion. then proceed to mass spect.

Question 9

2D-DIGE: stands for? what happens?

Answer 9

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.

Question 10

1D vs. 2D LC

Answer 10

1d: peptide mixture separated based on hydrophobicity. 2D-LC: isoelectric chromatogrphy followed by separation by hydrophobicity = MudPIT aka multidimensional protein identification technology

Question 11

mass spectrometry: 3 main parts

Answer 11

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.

Question 12

what does MALDI-TOF MS stand for

Answer 12

matrix assisted laser desorption ionization = MALDI. time of flight = TOF

Question 13

how does MALDI-TOF work

Answer 13

protein sample mixed with acidic matrix. UV laser causes protonation/ionization of peptides. proteins detach from matrix, and enter analyzer.

Question 14

how does analyzer separate proteins

Answer 14

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.

Question 15

ESI MS stands for?

Answer 15

electrospray inoization MS

Question 16

how does ESI MS work

Answer 16

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

Question 17

MS/MS stands for?

Answer 17

microsequency by tandem mass spectrometry

Question 18

what happens in MS/MS : select? fragment?

Answer 18

first mass analyzer selects ions of interest. CID, collision induced dissociation used to fragment selected ions by colliding them with gas (argon)

Question 19

MS/MS: second mass analyzer? how does fragmentation happen?

Answer 19

measures the fragment ions. occurs along the peptide backbone: each residue successively fragments off in N-C and C-N direction

Question 20

what are the ion fragments called

Answer 20

“Y” fragments (fragmenting from N terminal). from C terminal would be “b ions”

Question 21

difference between mass of ion fragments

Answer 21

is equal to the mass of one amino acid: use this to find out sequence

Question 22

2 techniques to map the neuroproteome?

Answer 22

voxelation and MALDI/MS imaging.

Question 23

voxelation?

Answer 23

slices of mouse brain cut into voxels (1mm3 cubes) and the extracted proteins subjected to LC-MS analysis.

Question 24

what happens in MALDI/MS imaging

Answer 24

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

Question 25

how can you analyze protein interactions?

Answer 25

functional proteomics: affinity column to isolate protein complex. SDS Page. excise bands and digest with trypsin. analyze with mass spect.

Question 26

clnical neuroproteomics involves?

Answer 26

analysis of protein differences between normal and diseased samples. can also analyse effects of specific drugs.

Question 27

HUPO BPP

Answer 27

human proteome organization’s brain proteome project

Question 28

example of study: 2 DE MS profiles of animals models of disease + normal aging found what?

Answer 28

common pathways dysfunctional in neurodegen diseases: oxidative phosphorylation and proteasome

Question 29

quantitative proteomics: how does it work

Answer 29

to see differences in protein quantity between conditions. label proteins (radioisotopes). mass spect: each peptide gives you two different peaks with two different masses. difference in mass will always be constant (will be = to C13 atoms). height of signal/intensity = relative abundance.

Question 30

SILAC

Answer 30

stable isotope labeling by amino acids in cell culture: give one culture C12, give one C13