When i have done mass by ESI MASS I got molecular weight with 18 less. Is it because of the OH group present in the tyrosine molecule?
Hi,
The situation you're describing not necessarily means that your peptide sequence contains a tyrosine residue, as there are several phenomena that result in a loss of 18 Da, such as peptide cyclization or the loss of water from serine or threonine. Are you sure it's 18 Da? Pyroglutamate formation at the N-terminus of the peptide results in a loss of 17 Da, for example.
The best way to determine the sequence of your peptide (and localize where the 18 Da is lost) would be to fragment it via MS/MS (or MSn), but I don't know if your mass spectrometer is capable of doing that.
Hope this helps, good luck!
Dear Eef Dirksen
I dont have serine and threonine in my peptide chain. Nor there is any cycization chances. what ive done is ive used fmoc asp(oll)-oh in my sequence after final amino acid coupling i have deprotected o allyl group. after neutraliztion of tetrakis ive taken little amount of resin cleaeved it and has submitted mass. one more thing fmoc group of last amino acid was not deportected. when i calculated i got 18 less. could u suggest what might be the reason
Dear Raghuvir if your mass spec result is consistently giving you loss of 18 Da not 1 7 Da, then it appears that one of the aspartic acid has cyclized to succinimide leading to loss of water. Trying doing MS/MS. If it has indeed formed succinimide then try to incubate your peptide in alkaline pH (8-9) under these conditions succinimide would be hydrolysed to give Asp/isoAsp. You can distinguish Asp from isoAsp by ETD-MS/MS.
Dear Sanjeev
Thanks for your valuable suggestion but the thing is i have two peptide sequences. one is 6 amino acid long and other is 9. but me not facing the same problem in 6 amino acid peptide chain though it contains both aspartic acid as well as tyrosine.
Succinimide formation is also effected by N+1 residue. If the aspartic acid is followed by a small residue. It has a higher propensity for succinimide formation. Do not worry about Tyr it could either be the modification of Asp or Asn to succinimide. Since you are getting loss of 18 Da and not 17 Da i assume it is the modifaication of one of the aspartic acid to succinimide. Do MS/MS on this peptide. Also incubate your peptide at high pH i am sure you will get back 18 Da after hydrolysis of succinimide.
Raghuvir, I was curious to know about your progress to find the reason of loss of 18 Da.
Could you find the position of loss of 18 Da?
Dear sanjeev
Not yet ill be happy to iinform you once i get out og this problem.
sanjeev, u were rite the problem i faced was due to succinimide formation. thank you very much for your valuable time.
Great!! Were you able to hydrolyse the succinimide at high pH?
What is the n+1 residue?
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