I want to quantify the recovery of tryptic peptides from a desalting method that I intend to use for preparing samples for LC-MS. My tryptic digests started out in ammonium bicarbonate and were eluted from my desalting columns with acetonitrile / 0.1% TFA and were then dried in a speedvac.
Can my dried peptides be reconstituted in ammonium bicarbonate so that they will be matrix matched with the starting material and also compatible with the BCA assay? I've read protocols where 0.1% trifluoroacetic acid or formic acid is used to reconstitute dried and desalted peptides prior to LC-MS analysis, but I think these acids will interfere with the BCA assay. Are they included specifically to prepare samples for LC-MS (which I am not yet doing at this point), or because they ensure that the dried peptides are thoroughly solubilized?
Thanks,
David
You can also use acetic acid, not sure if acetate interacts with the BSA reagents. They are used as a proton donor for ionization, they might also prevent electrostatic aggregation.
Dear David Shire ,
In the past I used TFA to freshly prepare a stock solution of my peptide where I first dissolve the peptides in trifluoroacetic acid (20 mg/mL) and subsequently evaporate off this solvent under a stream of N2.
See for example:
Article Tryptophan fluorescence study on the interaction of the sign...
You obtain a film of peptide that subsequently is dissolved in the appropriate solvent. And yes I would say you do this in order to make your peptide ready for solubilisation.
Best regards.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides