Indeed, proteins with disulfide bridge formation migrate faster in SDS gels than in their reduced form due to their denser packaging. If your protein(s) are purified you have the opportunity to cut the protein band separated by SDS-PAGE out of the gel, cleave it by trypsin and then perform a fingerprint by mass spectrometry. If both cysteines are not in close proximity to each other in their primary structure, you will get a (large) peptide fragment consisting of two single fragments connected by the disulfide bridge forming cysteines. In this way, you can identify exactly those cysteines that are involved in the formation of the putative disulfide bridge.

Indeed, proteins with disulfide bridge formation migrate faster in SDS gels than in their reduced form due to their denser packaging. If your protein(s) are purified you have the opportunity to cut the protein band separated by SDS-PAGE out of the gel, cleave it by trypsin and then perform a fingerprint by mass spectrometry. If both cysteines are not in close proximity to each other in their primary structure, you will get a (large) peptide fragment consisting of two single fragments connected by the disulfide bridge forming cysteines. In this way, you can identify exactly those cysteines that are involved in the formation of the putative disulfide bridge.

Hi there,

To make the distinction between free thiol and disulfide, you may first irreversibly alkylate free thiols (with AMS for instance) which results in heavier thiol side chain then reduce the disulfides with DTT and perform MS fingerprint of the final product to identify alkylated thiols and non alkylated thiols in the protein sequence.

Dear Elah

the formation of S-S bonds make the protein more compact and therefore a protein with SDS bonds migrate faster in SDS-page if not reduced by DTT.

In my blog ProteoCool (https://proteocool.blogspot.com/) on the presentation Overview of SDS-page you can see some examples of it.

Regarding your question: How identify which are the cysteines involved in S-S bonds with out perform the mutagenesis, a possible approach is trypsine digestion and mass spectrometry characterization of digested peptides

Article Direct mass spectrometric characterization of disulfide linkages

or as DOmique suggested, perform a modification with AMS or iodoacetammide of the free cisteines and analize than by MS.

if you are not in contact with a mass spectrometry group that can support you in this anaalisys, you can find several company that can provide this service

eg: https://www.alphalyse.com/post-translational-modifications/antibody-disulfide-bonds/

good luck

Manuele