Question 1

Which technique is specifically used to help determine the site of a disulfide bond in a protein?

Accepted Answer

Diagonal electrophoresis

Question 2

In diagonal electrophoresis, what is the expected migration pattern for a protein fragment containing an intrachain disulfide bond after the second dimension (after reduction)?

Accepted Answer

It migrates along the diagonal, indicating no change in size.

Question 3

Which of the following best describes the role of performic acid in diagonal electrophoresis?

Accepted Answer

It oxidizes disulfide bonds to cysteic acid, breaking the disulfide linkage.

Question 4

A protein is subjected to diagonal electrophoresis. After the first dimension, the protein migrates as a single spot. After reduction and the second dimension, two spots appear below the diagonal. What does this indicate?

Accepted Answer

The protein contains two interchain disulfide bonds.

Question 5

Which of the following statements about disulfide bonds is correct?

Accepted Answer

Disulfide bonds covalently link the R-groups of two cysteine residues.

Question 6

During diagonal electrophoresis, why do peptides with interchain disulfide bonds migrate off the diagonal after reduction?

Accepted Answer

Reduction causes the peptide fragments to become smaller, so they migrate differently in the second dimension.

Question 7

A researcher wants to determine if a protein’s disulfide bonds are interchain or intrachain. Which experimental result from diagonal electrophoresis would indicate the presence of only intrachain disulfide bonds?

Accepted Answer

All peptide fragments migrate along the diagonal after reduction.

Question 8

Which of the following best explains why diagonal electrophoresis is useful for mapping disulfide bonds in proteins?

Accepted Answer

It allows comparison of peptide fragment sizes before and after disulfide bond reduction.

Question 9

A protein with a single disulfide bond is mutated so that a serine is replaced by a cysteine, potentially creating a new disulfide bond. How could diagonal electrophoresis help determine if the disulfide pairing has changed?

Accepted Answer

By comparing the migration patterns of the wild-type and mutant proteins after reduction; a change in off-diagonal spots would indicate altered disulfide pairing.

Question 10

Which of the following is NOT a step in the diagonal electrophoresis workflow for mapping disulfide bonds?

Accepted Answer

Directly sequencing the protein without any separation.

Question 11

If a peptide fragment migrates along the diagonal in a diagonal electrophoresis experiment, what does this indicate about its disulfide bonds?

Accepted Answer

It contains no disulfide bonds or only intrachain disulfide bonds.

Question 12

Which chemical is commonly used to reduce disulfide bonds in proteins during diagonal electrophoresis?

Accepted Answer

Dithiothreitol (DTT)

Question 13

A protein is digested and subjected to diagonal electrophoresis. After reduction, a fragment migrates further in the second dimension than in the first. What does this suggest?

Accepted Answer

The fragment contained an interchain disulfide bond that was reduced, resulting in smaller fragments.

Question 14

Which of the following best describes the difference between interchain and intrachain disulfide bonds as revealed by diagonal electrophoresis?

Accepted Answer

Interchain disulfide bonds result in fragments migrating off the diagonal after reduction, while intrachain disulfide bonds do not.

Question 15

Which equation best represents the covalent linkage formed by a disulfide bond between two cysteine residues?

Accepted Answer

$$2 \mathrm{Cys} - \mathrm{SH} \rightarrow \mathrm{Cys} - \mathrm{S} - \mathrm{S} - \mathrm{Cys} + 2 \mathrm{H}^+$$

Question 16

A diagonal electrophoresis experiment is performed on a protein sample. After the second dimension, several spots are observed below the diagonal. What is the most likely explanation?

Accepted Answer

The protein contains interchain disulfide bonds that were reduced, resulting in smaller fragments.

Question 17

Which of the following steps is essential for identifying the specific location of a disulfide bond after diagonal electrophoresis?

Accepted Answer

Sequencing the off-diagonal peptide fragments.

Diagonal Electrophoresis and Disulfide Bond Analysis in Proteins

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