Question

A protein has the following sequence:   ALKJSCLKEPINWDVKLNCSLKRPILQMNLKCVGILKVN

The sequence contains three amino acids that can form a disulfide bond. However, only two of them actually make a disulfide bond in the protein. Propose a series of experiments that would allow you to determine between which two amino acids the disulfide bond occurs. Pick from: Salting out, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, SDS PAGE, affinity chromatography. HINT: do not overcomplicate the question.

Answer 1

Series of experiment for determination of position of occurence of disulfide bond will be:

1. Specific amino acid proteolysis- Prepare sample of given protein. These samples will have amino acid specific proteases. Since in this case three cysteine groups are present the proteases specific for methionine and glutamic acid will be taken.

2. Mass spectrometry- After proteolysis we will perform mass spectrometry which ionizes sample species and the ions are sorted based on masstocharge ratio. In the form of plot the results are given and the masses are determined.

Once mass spectrum is obtained it can be viewed to determine the position of the disulpfide bond.