Proteomics and CNS disease

Question 1

What is the study of omics looking at?

Answer 1

Looking at small things at a large scalee.gproteomics is looking at proteins in a large scale

Question 2

What is the proteome?

Answer 2

All the proteins which are in a sample at a given time under a set of conditions.

Question 3

What is proteomics analysis?

Answer 3

Is only ever a snap shot because protein levels/states vary according to cells/ tissues and are modified constantly according to environmental stimuli or developmental stage.

Question 4

Why are analysises of proteomics complex?

Answer 4

are all proteins detected? What are the proteins expressed? How sure are we? What is the expression levels? How sure are we?

Question 5

What are proteins?

Answer 5

Proteins are macromolecules made up of amino acids. The physical structure determines functions.

Question 6

How are proteins made?

Answer 6

In the Nucleus DNA is unwound to get a primary RNA transcript and mRNA. This is then taken to the cytosol where the mRNA is made into proteins. These modified proteins can then be studied using proteomics.

Question 7

Why study proteins?

Answer 7

Proteins do all the work in the cells and they are the mediators of the function of the cell. Any deviations could indicate a disease and proteins also make good drug targets for the cell.

Question 8

Which proteins should be looked at?

Answer 8

Select carefully but any that you want to study

Question 9

What is in the proteomic toolkit?

Answer 9

Gel based or liquid chromatography

Mass Spec

Database/algorithms

Software

Question 10

Why would you ise gel-based or liquid chromatography?

Answer 10

Simplify complex protein mixtures

Target specific proteins for analysis

Question 11

Why would you use mass spec?

Answer 11

Provide accurate molecular mass to charge measurements of intact proteins and peptides; create a unique spectra for a peptide

Question 12

Why would you use databse/algorithms?

Answer 12

Match the obtained MS spectra with specific known peptide spectra/protein sequences in databases to ID proteins.

Question 13

Why would you use software?

Answer 13

Determine how your protein interact in a given system.

Question 14

How do you extract and separate protein mixtures?

Answer 14

Put it in a buffer with detergents, reductants, denaturing agents of enzymes.

Question 15

How does gel electrophoresis work?

Answer 15

Running proteins through a gel which will be seperated by charge. Once they reach their isoelectric point they will stop moving and can be stained.

They are then placed on another gel and seperated on weight.

Software can then identify any proteins which are the same and intensity can then be measured.

Question 16

What are difficulties with 2D gels?

Answer 16

Reproducibility is hard as these take hours to do one
Small dynamic range - Stains can be hard as the more abundant proteins are easily seen whilst less
Co-migrating spots form complex regions, multiple proteins in one spot
Streaking and smearing
Weak spots and background

Question 17

What would you do after getting your 2D gel results?

Answer 17

Cut out spots of the gel you want to look at, add trypsin to cut the proteins up and use PCR to elongate them. You can then compare the protein to other proteins in a database.

Question 18

Is there a database for peptide and protein identification?

Answer 18

Yes

Question 19

What is shotgun proteomics in the brain?

Answer 19

Tissue from brain taken and split into peptides using trypdin. The residual detergent molecules attached to them are wahed off using silica cation exchange. Once clean they can go through the LC/MS machine to give you a spectrum which can be compared to others in a database.

Question 20

What are the advantages of shotgun proteomics?

Answer 20

Quantitative shotgun proteomics

Clearer quantitative comparisons between isotopes tags samples. Ratios of light and heavy isotopes allow detection of differences.

Question 21

Disadvantages of shotgun proteomics?

Answer 21

Expensive
Labour intensive and time consuming
Not every protein will get labelled
More protein is needed

Question 22

What is label free quantification?

Answer 22

Protein levels are determined based on the intensity of the peptide peak

Question 23

What are advantages of label-free quantification?

Answer 23

No labelling step required

Much larger coverage of proteins

No expensive reagents for labelling

Very little proteins needed

Question 24

What are disadvantages of lable-free quantification?

Answer 24

Variability can be high

A single sample run can take 2 hours

Large studies are hard

Question 25

What are the results of label-free quantification?

Answer 25

You get a long list of proteins which change rank based on their probability of coming.

Question 26

What are the applications of 2d Gel proteomics on cell death studies?

Answer 26

Can look at proteins before and after a stroke and narrow down cells which are on the cusp of dying to see if they can be stopped. They could not.

Question 27

Example - apolipoprotein genotype in alzheimers disease?

Answer 27

People with copies of apolipoprotein are more at risk of developingalzheimerswith 40% of those suffering with ApolE4.So they looked into APOlE4 by getting wildtype mice and APOE4 mice - they found the mitochondrial proteins were different and were modulated differently.

Question 28

How did they use proteomics to look at the human brain?

Answer 28

They looked at Glioblastoma proteins (this is hard as the cells are dead so they had to find live bits). They compared mitochondrial proteins and found that the mitochondria could regulate GBM which could help with treatment.

Also found there was a downregulation of oxidative phosphorylation respiratory chain.

Question 29

What would you do with post proteomics?

Answer 29

Help you make new hypothesizes to test.