Chapter 3: 3.1 Proteomics

Question 1

What questions does proteomics allow researchers to ask? (8)

Answer 1

1. Which proteins are present in an organism, tissue, cell, organelle, etc.?
2. How much of a certain protein is present?
3. Where is the protein located?
4. Are there any protein variants present (isoforms from alternative splicing, post-translationally modified proteins, etc.)?
5. In a protein complex - which proteins are present and how do they interact?
6. When the state of a cell changes, do the proteins expressed change? Do the amounts of proteins change?
7. Can protein changes be used to diagnose diseases?
8. Which proteins can be used as drug targets in certain diseases?

Question 2

Describe the process of:

High-throughput LC-MS/MS

Answer 2

1. A complex mixture of proteins is difested by a protease
2. Liquid chromatography (LC) separates the proteins into less complex fractions
3. The fractions are injected by electrospray ionization into a tandem mass spectrometer
4. Multiple MS/MS cycles until all the proteins peptide sequences have been determined

Question 3

What does “LC” stand for in “High-throughput LC-MS/MS”?

Answer 3

Liquid chromatography

Question 4

State:

The Pros of High-throughput LC-MS/MS

Answer 4

Allows taking a mixture of organelles, break them open to:
1. Release proteins
2. Identify proteins
3. Figure out organelle of origin of proteins

Can identify locations of hundreds of proteins SIMULTANEOUSLY

Question 5

State:

The Cons of High-throughput LC-MS/MS

Answer 5

Sensitivity needs to be improved
* Requires a lot of cells to use LC-MS/MS (200,000-700,000 cells)

Question 6

Define:

Phosphoproteomics

Answer 6

The identification and quantification of phosphorylation sites on proteins

Question 7

1. What are proteins phosphorylated by?
2. What are proteins dephosphorylated by?

Answer 7

1. Kinases
2. Phosphatases

Question 8

What can protein phosphorylation/dephosphorylation affect?

Answer 8

Affect the function/interactions of the proteins

Question 9

State:

The Pros of Phosphoproteomics

Answer 9

Provides insight into how phosphorylation can change protein interactions and function

Question 10

State:

The Cons of Phosphoproteomics

Answer 10

Need a lot more cells (50 - 100x) more than LC-MS/MS

Question 11

Why does phosphoproteomics require that many cells?

Answer 11

Only a small fraction of the proteins will be phosphorylated

Question 12

In Phosphoproteomics:

How are the phosphorylated and dephosphorylated proteins separated? When?

Answer 12

Researchers usually have to use affinity chromatography
* Before LC-MS/MS