Proteomics

Question 1

Define proteomics

Answer 1

The study of the entire complement of proteins, how they’re modified, when and where they’re expressed, how they’re involved in metabolic pathways and how they interact with one another

Question 2

What are proteins made up of?

Answer 2

Amino acids

Question 3

What is special about proteins?

Answer 3

They all have distinctive molecular weights

Question 4

Give an example of an enzymatic catalysis protein

Answer 4

p450

Question 5

Give an example of a transport and storage protein

Answer 5

Haemoglobin

Question 6

Give an example of a coordinated motion protein

Answer 6

Muscle proteins

Question 7

Give an example of an immune protection protein

Answer 7

Antibodies

Question 8

Give examples of proteins involved in the generation and transmission of nerve impulses

Answer 8

Rhodopsin and acetylcholine

Question 9

Give an example of a protein involved in the control of growth and differentitation

Answer 9

Growth factor proteins

Question 10

What are the two proteomics approaches?

Answer 10

Top down and bottom up

Question 11

Briefly describe the bottom-up proteomics approach

Answer 11

1. Isolate proteins
2. Reduce and alkylate
3. Digest
4. Separate out peptides
5. Mass determine peptides and sequences
6. Informally collate all of the data to determine what the protein is and how much there is

Question 12

Proteins are structurally complicated structures, true or false?

Answer 12

True

Question 13

How can you break the four levels of protein structure?

Answer 13

By trypsin digest

Question 14

Give an example of a reducing agent, how it works, and what the outcome is for proteins

Answer 14

Dithiothreitol (DTT)

Disrupts disulphide bonds to free thiol groups

Question 15

Give an example of an alkylating agent, how it works, and what the outcome is for proteins

Answer 15

Iodoacetamide (IAA)
Maintains disrupted disulphide bonds by alkylating cysteine groups and thus preventing them from reforming disulphide bonds
Causes the formation of S-carbamidomethyl cysteine groups

Question 16

What do digestive enzymes do?

Answer 16

Recognise specific amino acids and cleaves at this specific site only

Question 17

What is the most commonly used digestive enzyme?

Answer 17

Trypsin

Question 18

Where does trypsin cut?

Answer 18

At the C-terminus of lysine and arginine residues (except when either is followed by proline)

Question 19

What sorts of peptides will be excluded from digestion analyses?

Answer 19

Very short and very long peptides

Question 20

What do you need to do in order to maximise the liberation of tryptic peptides?

Answer 20

Need to disrupt the 3D structure of proteins

Question 21

What is dithiothreitol (DTT) used for?

Answer 21

To reduce/disrupt disulphide bonds formed between cysteine groups

Question 22

What is iodoacetamide (IAA) used for?

Answer 22

To alkylate cysteine groups to prevent the reformation of disulphide bonds

Question 23

What is trypsin used for?

Answer 23

To generate MS amenable peptides

Question 24

What do samples that are a mixture of peptides have a range of?

Answer 24

pI
Hydrophobicity
Size

Question 25

What is utilised to establish a separation gradient on HPLC?

Answer 25

The wide range of hydrophobicities

Question 26

As peptides elute from LC column what are they subject to?

Answer 26

Vaporisation, ionisation and detection by the mass spectrometer

Question 27

What are ionised peptides subject to?

Answer 27

Tandem mass spectrometry to determine amino acid sequence

Question 28

What does LC stand for?

Answer 28

Liquid chromatography

Question 29

What is liquid chromatography similar to?

Answer 29

Paper chromatography

Question 30

Liquid chromatography separates out molecules according to their what?

Answer 30

Hydrophobicity