How can I arrange peptides with S-S bonds in order?

[TytoAlba95]

Homework Statement

A 30 residue peptide was treated with trypsin and the tryptic peptides we're separated by HPLC. Four peakes were obtained. Peptides corresponding to A, B, C and D we're reduced and alkylated selectively at Cys residues.
The sequences obtained are :

Relevant Equations

Please refer to the image below:

I can't solve this at all. Please suggest some clues.

 

[TeethWhitener]

Do you understand the importance of the reduction/alkylation at cysteine residues? Note that this step happened after the HPLC separation.

 

[TytoAlba95]

Do you understand the importance of the reduction/alkylation at cysteine residues? Note that this step happened after the HPLC separation.

Thanks for pointing that out. I have read COFRADIC and in normal COFRADIC these modifications occur before the HPLC, isn't it?
The aim of reduction is to remove inter/ intra-chain disulphide bonds (here intra-chain) and the purpose of alkylation is to block the reactive -SH groups to prevent further s-s bonding.

 

[TeethWhitener]

Thanks for pointing that out. I have read COFRADIC and in normal COFRADIC these modifications occur before the HPLC, isn't it?
The aim of reduction is to remove inter/ intra-chain disulphide bonds (here intra-chain) and the purpose of alkylation is to block the reactive -SH groups to prevent further s-s bonding.

COFRADIC isn’t relevant to this problem (unless the question says otherwise), but yes, there are protocols where the S-S bonds are reduced and protected first.

Focusing on the problem, you know that the HPLC is performed before the disulfide bridges are cleaved and protected, so given that info, which of the four sequences (with disulfide linkages) from your original problem are possible?

 

[TytoAlba95]

Focusing on the problem, you know that the HPLC is performed before the disulfide bridges are cleaved and protected, so given that info, which of the four sequences (with disulfide linkages) from your original problem are possible?

I can only guess, (d)? I did some research but couldn't find any info on intra-chain S-S bonding pattern.

However the answer provided is (c).

 

[TeethWhitener]

I can only guess, (d)? I did some research but couldn't find any info on intra-chain S-S bonding pattern.

However the answer provided is (c).

Don’t guess. It might help to break the listed sequences into the fragments from HPLC and see where the cysteine linkages are.

 

[TytoAlba95]

I'm sorry, I can't come to any conclusion. I'm stuck. To me (b) looks too complicated to exist, and the rest three seem feasible.

 

[TeethWhitener]

Did you try what I said? You need to put in some effort before I can give you more help.

 

[TytoAlba95]

Did you try what I said? You need to put in some effort before I can give you more help.

I couldn't understand initially but on a second attempt I understood! The peptide fragments that were linked by S-S bond were eluted together. The answer is c.