I am doing an HDX experiment. Protein contains two cysteine residues. Upon desalting (removal of DTT and other salts using water containing 0.1 % formic acid at pH ~2.8 ) of protein, it forms a disulphide bond and hence, that part is not digested well by the pepsin.
I am thinking to keep the protein in the reduced form by adding the TCEP (because it can work at low pH) during online digestion. Can any one tell me that whether the presence of TCEP in digestion buffer will affect my immobilized pepsin column (from applied biosystem) or not?
Since it is an HDX experiment, it should be done quickly. Therefore, I don't want to add an another step as for alkylation.
Thanks for your time for going through my question.
Hello Prashant,
Pepsin is known to work in the presence of TCEP both online and offline. So, you should be able to carry out online pepsin digestion in the presence of TCEP.
You can get more information about it in these paper:
Article Simultaneous Reduction and Digestion of Proteins with Disulf...
Article Accessing the Reproducibility and Specificity of Pepsin and ...
It might reduce the life of pepsin column though. Cleaning your pepsin column with TCEP-free buffer at the end of your experiment should be useful.
I hope that helps!
Good-luck for your experiment!
Jogender
Thanks Jogender Singh.
You suggestion really helped and improved my pep map profile.
thanks
I am glad that it helped in improving the peptide map profile, Prashant!
Cheers!
- Badges
- Science method