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…
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:
Select an option to attempt
Correct Answer: Option D
Explanation
Trypsin digestion and missing bands on SDS-PAGE:Trypsin cleaves after Lys (K) or Arg (R), EXCEPT when followed by Proline (P).Given: 10 Lys + 2 Arg = 12 potential cleavage sites → 13 expected fragments.Observed: only 10 bands → 3 fewer cuts occurred. Possible reasons:A. Buried Lys/Arg: inaccessible to trypsin in native protein — but in SDS-PAGE, the protein is fully denatured; however, the DIGESTION was done on the native protein potentially, or it's a distracting option. Actually, if buried, trypsin cannot access them.B. Arg/Lys before Pro: trypsin does NOT cleave Lys-Pro or Arg-Pro bonds — well-established rule ✔C. Lys/Arg before negatively charged residue: trypsin is somewhat less efficient but generally still cleaves — this is NOT a primary reason for missed cleavages.D. Modified Lys ε-N (e.g., acetylation or methylation of the ε-amino group): removes the positive charge, making the site unrecognised by trypsin ✔E. C-terminal Lys or Arg: produces no extra fragment (the last residue at the C-terminus doesn't create an additional cut) — this reduces expected count by 1 ✔Most valid reasons: B, D, E (Option 4 = A, B, D and E).Answer: A, B, D and E (Option 4).