Characterizing Protein Properties

Question 1

What are the four characteristics to look at for protein characterization? What are the characterization methods for each characteristic?

Answer 1

Question 2

How does SDS-PAGE characterize proteins? What characteristic does it characterize.

Answer 2

Size.
Note: Opposite of gel-filtration where bigger proteins move through gel quicker.
Note 2: Only measures denatured proteins, not full complexes!

Question 3

How does Mass Spectronomy characterize proteins? What characteristic does it characterize.

Answer 3

Amino Acid sequence.

Question 4

Mass spectrometry can only be used for proteins of about 40 amino acids or smaller. How can we still use it?

Answer 4

Ignore purple and just focus on blue.
Be able to explain why we need two differently cut strands for the same sequence.
Use different proteases to make different breaks in the sequence!

Question 5

Define protease. What’s the one exception to proteases?

Answer 5

Proteases break a peptide bond based on the identities of the adjacent amino acids. (They break proteins)
Exception: proteases will not cleave a bond when the “other” residue is proline.

Question 6

Trypsin is an enzyme, what does it do? What is the enzyme classification?

Answer 6

Cleaves when R(n-1) is Arg or Lys (positive amino acid!) and Rn is NOT proline.

Question 7

Chymotrypsin is an enzyme, what does it do? What is the classification of enzyme?

Answer 7

Cleaves when R(n-1) is Phe, Trp, Tyr, or Leu (bulky and aromatic!)
BUT not when Rn is Proline.

Question 8

What is notable about protease specificity?

Answer 8

They break peptide bonds based on the identities of either of the adjacent amino acids(not both).

Question 9

Define Scissile peptide bond

Answer 9

Peptide Bond broken by protease.

Question 10

R(n-1) is which residue? R-CH-CO- or NH-CH-R

Answer 10

R-CH-CO-

Question 11

R(n) is which residue? R-CH-CO- or NH-CH-R

Answer 11

NH-CH-R

Question 12

What are 2 things Mass Spectrometry techniques can do ?

Answer 12

1. Help determine mass of protein with small amount of protein, quick experiment, and very accurately.
2. Able to sequence amino acids about up to 40 amino acids in length.

Question 13

Explain how Electron Spray Ionization Mass Spectrometry (ESI) works.

Answer 13

The data from mass spectrometry is sufficient to determine the mass of the original molecule.

Question 14

Explain how tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein works.

Answer 14

Question 15

Explain how tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein works.

Answer 15

Question 16

Explain how tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein works.

Answer 16

Question 17

Explain how tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein works.

Answer 17