MODULE 4: Chapter 5.2

Question 1

What role does genomics play in protein characterization?

Answer 1

Genomics helps characterize the structure and function of individual proteins through the expression of cloned gene sequences.

Question 2

What is the primary biochemical method for identifying an unknown purified protein?

Answer 2

Determining its amino acid sequence biochemically.

Question 3

What technique is commonly used for amino acid sequence analysis in proteomic methods?

Answer 3

2-D PAGE followed by amino acid sequence analysis.

Question 4

Who developed the Edman degradation method?

Answer 4

Pehr Edman, a Swedish biochemist.

Question 5

What reagent was originally used in Sanger’s protein sequencing method?

Answer 5

1-fluoro-2,4-dinitrobenzene.

Question 6

What is Edman degradation primarily used for?

Answer 6

To deduce the amino acid sequence of short polypeptides.

Question 7

What is the advantage of Edman degradation over Sanger’s method?

Answer 7

It does not require the input of additional protein after each round of cleavage.

Question 8

What is the maximum length of peptides that can be sequenced using Edman degradation?

Answer 8

Up to 50 amino acid residues.

Question 9

What types of protease enzymes are used to cleave proteins for Edman degradation?

Answer 9

• Trypsin
• Chymotrypsin

Question 10

What does trypsin cleave on?

Answer 10

The carboxyl side of lysine and arginine residues.

Question 11

What is tandem mass spectrometry used for?

Answer 11

To determine the amino acid composition of peptide fragments.

Question 12

What are the two ionization techniques mentioned for mass spectrometry?

Answer 12

• Electrospray ionization (ESI)
• Matrix-assisted laser desorption/ionization (MALDI)

Question 13

What is the basic principle behind mass spectrometry?

Answer 13

It measures the mass-to-charge ratio (m/z) of molecules.

Question 14

What is solid-phase peptide synthesis used for?

Answer 14

To synthesize peptide antigens for antibody production and peptide-based therapeutic drugs.

Question 15

Who developed the strategy for solid-phase peptide synthesis?

Answer 15

Bruce Merrifield.

Question 16

What is the direction of synthesis in solid-phase peptide synthesis?

Answer 16

From the C terminus to the N terminus.

Question 17

What is the first step in solid-phase peptide synthesis?

Answer 17

Attaching the C-terminal amino acid to a resin molecule.

Question 18

What is Fmoc in the context of solid-phase peptide synthesis?

Answer 18

A blocking group (9-fluorenylmethoxycarbonyl) used on the N-terminal side of incoming amino acids.

Question 19

What happens to the protecting groups in solid-phase peptide synthesis?

Answer 19

They are removed after the peptide synthesis is complete.

Question 20

What is the typical length of peptides generated through solid-phase peptide synthesis?

Answer 20

15 to 25 amino acid residues.

Question 21

True or False: Edman degradation can efficiently sequence long polypeptides without cleavage.

Answer 21

False.

Question 22

Fill in the blank: The Edman degradation method provides protein sequence information based on chemical determinations of _______.

Answer 22

[N-terminal amino acids]

Question 23

What is the significance of the 1958 Nobel Prize awarded to Frederick Sanger?

Answer 23

It was for determining the amino acid sequence of insulin.

Question 24

What does the process of mass spectrometry involve measuring?

Answer 24

The mass-to-charge ratio (m/z) of molecules.

Question 25

What are tryptic fragments?

Answer 25

Peptide fragments produced by trypsin cleavage.

Question 26

What is Edman degradation?

Answer 26

A protein sequencing method based on labeling and cleaving the N-terminal residue without disrupting the rest of the polypeptide chain. ## Footnote Edman degradation allows for the sequencing of proteins from the amino-terminal end.

Question 27

What is the main advantage of mass spectrometry over Edman degradation for protein identification?

Answer 27

Complex mixtures of protein subfragments can be analyzed without prior purification. ## Footnote This significantly reduces sample preparation time compared to Edman degradation.

Question 28

What is a disadvantage of Edman degradation?

Answer 28

It only sequences proteins from the amino-terminal end and covers a small region of the total protein sequence. ## Footnote This limits its overall effectiveness in determining the full sequence of large proteins.

Question 29

What does mass spectrometry use to identify proteins?

Answer 29

Mass analysis of peptide fragments and computer algorithms to calculate probabilities. ## Footnote This method relies on comparing unknown proteins to predicted protein sequences from a genomic database.

Question 30

What is trypsin?

Answer 30

A protease that cleaves a polypeptide chain on the carboxyl side of lysine or arginine residues. ## Footnote Trypsin is commonly used in protein digestion protocols.

Question 31

What is chymotrypsin?

Answer 31

A protease that cleaves a polypeptide chain on the carboxyl side of tyrosine, tryptophan, and phenylalanine residues. ## Footnote Chymotrypsin helps provide shorter polypeptide chains suitable for Edman degradation.

Question 32

What does electrospray ionization (ESI) do?

Answer 32

Prepares proteins for mass spectrometry by providing highly charged peptide ions in the gas phase. ## Footnote ESI is a key step in the mass spectrometry process.

Question 33

What is matrix-assisted laser desorption/ionization (MALDI)?

Answer 33

A method for generating peptide ions for mass spectrometry that uses a laser to ionize the peptide fragments. ## Footnote MALDI is an alternative ionization method used in mass spectrometry.

Question 34

What is solid-phase peptide synthesis?

Answer 34

A technique to synthesize peptide antigens for antibody production and to manufacture peptide-based therapeutic drugs. ## Footnote This method is widely used in the production of therapeutic peptides.

Question 35

What would happen if the Fmoc protecting group was not added to amino acid 2 during peptide synthesis?

Answer 35

The product(s) after the first round of coupling would include unprotected amino acid 2. ## Footnote This could lead to unwanted side reactions during subsequent coupling steps.

Question 36

True or False: Mass spectrometry requires access to a genomic sequence database for protein identification.

Answer 36

True. ## Footnote Without a genomic database, mass spectrometry cannot effectively identify proteins.