64. A protein with 10 lysines and 2 arginine residues was digested with trypsin and the peptide
mixture was resolved on an SDS-PAGE. After silver staining, 10 bands were detected instead of 13.
Select the plausible reasons from the following:
A. Some of the Arginines and Lysines were inside the protein core.
B. Some of the Arginines and Lysines were followed by a Proline residue.
C. Some of the Arginines and Lysines were followed by a negatively charged residue.
D. Some of the Lysines were modified at the epsilon Nitrogen atom.
E. One of the Lysine or Arginine was at the extreme C-terminus.
Choose the correct answer from the options given below:
A. A, B, C and D.
B. A, B, C and E.
C. B, C, D and E.
D. A, B, D and E

Introduction:

When performing trypsin digestion on proteins for peptide mapping, the expected result is that trypsin will cleave the protein at lysine (K) and arginine (R) residues, unless these residues are followed by proline (P) or other factors that inhibit cleavage. In this case, after silver staining of the SDS-PAGE gel, 10 bands were detected instead of the expected 13, which suggests that some of the lysines and arginines did not undergo cleavage. Several factors could explain this discrepancy, and understanding these can help in interpreting the results of such protein analysis experiments.

Factors Affecting Trypsin Cleavage:

Several conditions might affect the ability of trypsin to cleave the peptide chain at lysines and arginines:

• A. Some of the Arginines and Lysines were inside the protein core.

  • True. If some lysines and arginines are buried within the protein core and not exposed on the surface, they might not be accessible to trypsin, preventing cleavage at these residues. This would result in fewer bands after digestion.

• B. Some of the Arginines and Lysines were followed by a Proline residue.

  • True. Proline is a known inhibitor of trypsin cleavage. If lysines or arginines are followed by proline, trypsin cannot cleave at these residues, leading to fewer peptide fragments. This is a common phenomenon in peptide mapping experiments.

• C. Some of the Arginines and Lysines were followed by a negatively charged residue.

  • False. The presence of negatively charged residues following lysine or arginine does not typically inhibit trypsin cleavage. Trypsin preferentially cleaves after lysine and arginine regardless of the charge of the following residue, so this condition is unlikely to explain the observation.

• D. Some of the Lysines were modified at the epsilon Nitrogen atom.

  • True. Post-translational modifications, such as acetylation or methylation at the epsilon nitrogen of lysine, can block trypsin cleavage. This would prevent the cleavage of those lysines, leading to fewer bands.

• E. One of the Lysine or Arginine was at the extreme C-terminus.

  • True. Trypsin usually does not cleave at the C-terminal end of the protein. If a lysine or arginine is located at the C-terminus, no further cleavage can occur beyond that point, potentially reducing the number of observable peptide fragments.

Correct Answer:

The plausible reasons for the reduction in the number of peptide bands detected on SDS-PAGE are:

• A. Some of the Arginines and Lysines were inside the protein core.

• B. Some of the Arginines and Lysines were followed by a Proline residue.

• D. Some of the Lysines were modified at the epsilon Nitrogen atom.

• E. One of the Lysine or Arginine was at the extreme C-terminus.

Thus, the correct answer is: D. A, B, D, and E

Conclusion:

In trypsin digestion experiments, several factors such as the accessibility of residues, the presence of proline, post-translational modifications, and the position of residues (e.g., C-terminal) can influence the cleavage pattern and the number of peptide fragments produced. By understanding these factors, researchers can better interpret the results of their SDS-PAGE analysis and adjust experimental conditions for optimal peptide mapping.