I have synthesized a di-peptide using Fmoc chemistry and cleavage was done using concentrated TFA alone as no side-chain protection containing amino acid is involved in my sequence. During characterization process, i have got a single peak in HPLC but when i have done ESI-MS of the HPLC elution the observed mass was 3 less than the calculated mass. I have face the same problem when i have repeated the synthesis. Can anyone explain to me the possible cause of getting lesser observed mass then the calculated mass?
Mr. Saikia,
The ESI-MSs of oligopeptides (di- to deca-) show formation of Na+/NH4+-adducts of [M], dimeric associates and/or cyclic products. These are data of about 250-300 peptides, their complexes and anionic/cationic forms in our database of datablocks. You could account for a possible cyclization in GP.
Could you upload a chromatogram and ESI- and CID MS2 spectrum of your compound and/or a *.raw file?
Hi Jahnu,
a mass difference of 3 sounds very strange for me, I never saw a deprotonation in LC-MS since the mobile phase pH is acidic (plus: this would not give a signal in positive mode MS, since the net charge changes to negative). Are you sure with the sequence? Any miscalculation? C terminus Amide or Carboxyl? Right AA used for coupling (e.g. glutamine and glutamic acid mass difference 1)? Positive or negative mode MS settings?
I attached some links and an excel file where you may find further information for your problem.
https://www.researchgate.net/post/What_may_be_the_reason_if_I_am_getting_higher_mass_calculated_mass_42060_experimentally_447_in_mass_spectrometry
https://www.researchgate.net/post/Has_anyone_seen_LC-MS_contamination_of_mass_117_212_and_233_in_ESI_negative_and_156_in_ESI_positive
Dipeptides especially before neutralization using Fmoc chemistry are prone to cyclize and form diketopiperazines (DKP's) this would give loss of water M-18 for the mass. Addition of an oxygen (oxo group) a in place of 2 hydrogen's somewhere in the DKP could give an M+H ion with a 3 amu mass deficit from the free dipeptide. Could this be what is happening since you did not use any reducing agent or free radical scavengers in your TFA deprotection step? Did you exclude oxygen (or air) from the TFA cleavage reaction?
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