During sample preparation for mass spectrometry, disulfide bonds are reduced with DTT followed by alkylation of the free thiol group on cysteines.
But if your protein of interest does not contain any cysteines at all, is there a point in doing this, or can this step safely be skipped in this case?
Hi Alexander,
Breaking the disulfide bonds would largely facilitate the efficiency of trypsin digestion (through disrupting the protein conformation). But if it's not a concern, you could possibly skip the DTT and IAA incubation.
IMO, a given sample could still be identified w/o reduction and alkylation. Apart from that, other non-cysteine containing peptides could also aid the identification.
Maybe you should test this in a trial analysis.
Kay
The reduction and alkylation steps are unnecessary for these proteins that do not contain disulfide bonds.
Take β-casein as an example, the reduction and alkylation steps are always skipped in numerous papers.
Agreed I try and not do IAA treatment whenever possible.
If you have no cysteines there is not point.
For general archaea and bacterial proteins generally have much less cysteines than human or plant proteins.
This for bacterial proteomes very often I do double reduction with no IAA. reduce before trypsin digestion and after trypsin digestion, go straight into de-salt at low pH and random disulfide bonds should not reform
Hi Alex,
I have analyzed SDS-PAGE protein bands without reduction and alkylation. I forgot the step and I was freaking out till I did MALDI. Surprisingly, I have ID of all the bands with very strong score. So I believe the reduction and alkylation of proteins aid in protein conformations and definitely help in analyzing data however that step is not mandatory. The good thing in your case is you know your protein has no cysteine, then why to add extra step?
Hope this helps.
Indeed, if the protein does not contain any Cys residues you can skip this reduction/alkylation step.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics