Peptide Sequencing

Question 1

What CHEMICAL method can be used to determine primary protein structure?

Answer 1

Edman Degradation

Question 2

What PHYSICAL method can be used to determine primary protein structure?

Answer 2

Mass Spectrometry

Question 3

What BIOLOGICAL methods can be used to determine primary protein structure?

Answer 3

• Dideoxynucleotide Sequencing
• Next Generation Sequencing (NGS)

Question 4

During Polypeptide Cleavage in Chemical Sequencing, what amino acid does Cyanogen Bromide (BrCN) cleave?

Answer 4

Cleaves at carboxyl side of Met

Question 5

Which TWO amino acids is Trypsin selective to?

Answer 5

Carboxyl side of:
* Arg (R)
* Lys (K)

(+ve basic AAs)

Question 6

Which THREE amino acids is Chymotrypsin selective to?

Answer 6

Carboxyl end of:
* Tyr (Y)
* Phe (F)
* Trp (W)

(aromatic AAs)

Question 7

Which FOUR amino acids is Pepsin selective to?

Answer 7

• His
• Gly
• Glu
• Gln

Question 8

What are the FOUR steps in the cleavage mechanism for Chymotryspsin?

Answer 8

1. Substrate binding and alignment in the active site
2. Nucleophilic attack of the peptide bond (acyl-enzyme formation)
3. Product 1 released
4. Hydrolysis of acyl-enzyme and release of product 2

Question 9

What are Steps 1 and 2 of Edman Degradation?

Answer 9

1. Specific Cleavage of protein
2. Purify Peptides by RP-HPLC

Question 10

What are the steps for Step 3 (Stepwise Degradation with PITC) of Edman Degradation?

Answer 10

1. First reaction at basic pH
2. Cleavage and conversion into PTH in anhydrous trifluoroacetic acid
3. Derivatised amino acid is extracted by organic solvent

Question 11

How are PTH AAs identified in Step 4 of Edman Degradation?

Answer 11

Identified by C18 RP-HPLC

Question 12

What is Step 5 of Edman Degradation?

Answer 12

Alignment of Peptides

Question 13

How many AAs can be sequenced directly with Edman Degradation?

Answer 13

Up to 50

Question 14

What bonds are reduced in Edman Degradation?

Answer 14

Disulfide bonds

Question 15

Which peptides/proteins cannot be sequenced in Edman Degradation?

Answer 15

N-modified

Question 16

What is the accuracy of Edman Degradation?

Answer 16

98%

Question 17

What is chemical in Sanger’s Reagent?

Answer 17

FDNB

Question 18

What does Sanger’s Reagent do to the AA?

Answer 18

Modification of N-terminal

Question 19

What does Sanger’s Reagent do to the polypeptide?

Answer 19

Hydrolysis of the polypeptide

Question 20

What do Mass Spectrometers measure?

Answer 20

Mass / Charge ratio of an ion

Question 21

How are proteins physically sequenced and identified by Mass Spectrometry?

Answer 21

Peptide Mass Fingerprinting (PMF)

Question 22

What do peptides fragments yield in predictable ways in Mass Spectrometry?

Answer 22

Daughter Ions

Question 23

What are the FOUR comparisons between Mass Spectrometry vs Edman Degradation?

Answer 23

• More sensitive
• Can fragment peptides faster
• Does not require proteins to be purified
• No problem identifying modified proteins