SFP5 - Protein characterisation and primary structure determination

Question 1

What is a homotetramer?

Answer 1

A protein complex consisting of four identical subunits

Question 2

What method is used to determine native molecular weight?

Answer 2

Gel filtration chromatography / size exclusion chromatography

Question 3

How does it work?

Answer 3

Proteins pass through the beads in the column. Smaller proteins require more elution volume to be removed. The elution volume can then be compared to the elution profile of the protein to that of known molecular weight standards

Question 4

What is meant by elution?

Answer 4

Elution refers to the process of washing or flushing a substance out of a column or other stationary phase by running a mobile phase through it

Question 5

What is meant by elution volume?

Answer 5

Elution volume refers to the volume of mobile phase required to elute a substance from the column

Question 6

What does SDS do?

Answer 6

Coats the protein in a negative charge in proportion to their size

Question 7

How can the molecular weight of a denatured protein be determined?

Answer 7

SDS coated proteins are placed onto an acrylamide gel with small pores in it. Then an electric current is supplied which forces the proteins towards the positive electrode at different speeds. The molecular weight of a denatured protein can be estimated based on its migration distance through the gel compared to the migration distances of known molecular weight standards that are run on the same gel.

Question 8

Identical subunits can be determined by the use of native mwt and denatured mwt. What is the equation?

Answer 8

native mwt / denatured mwt

Question 9

What two methods can be used for the determination of the primary structure?

Answer 9

1. Edman degradation
2. Mass spectrometry
   A pure protein is required from SDS page / 2D gel

Question 10

What is the first step in protein identification by MS?

Answer 10

2D gel electrophoresis or SDS page

Question 11

What is the first step in Peptide Mass Fingerprinting (PMF)?

Answer 11

Cut out the protein of interest (spot / band from gel)

Question 12

Second step?

Answer 12

Proteolytically digest in gel with trypsin

Question 13

Where does trypsin cut the protein?

Answer 13

At K (lysine) and R (arginine) towards the c-terminus

Question 14

So, why would this make the peptides unique?

Answer 14

Each peptide from the parent protein would be unique because peptides can only be cut at K and R. So each protein has a specific order at where they are at. These act as a fingerprint to the protein of interest.

Question 15

What is the third step?

Answer 15

Use mass spectrometry to determine the mass values of each peptide.

Question 16

How is this carried out?

Answer 16

Protonate them (artificially charge them) and measure their m/z values

Question 17

How can the mass values of the peptides be used?

Answer 17

Compare them to a sequence database. They have been derived from gene mapping. That is why protemoics is referred to as a post-genomic technology.

Question 18

Our protein can have its peptides compared to a known protein from the database and be determined.

Question 19

What is a downfall of PMF?

Answer 19

If the genomic data is poor or incomplete, it is less effective

Question 20

What is the first step of tandem MS (MS/MS)?

Answer 20

Generate tryptic peptides like PMF

Question 21

What is the second step of MS/MS?

Answer 21

A collision cell selects ionised peptides and fragments them further