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Topic: mass spectrum of tritium labelled peptides?  (Read 7348 times)

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Offline msd

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mass spectrum of tritium labelled peptides?
« on: November 19, 2012, 09:32:57 AM »
I have some questions regarding tritium labelled peptides, some uncertainties I have and would greatly help me if I could talk with somebody with more knowledge in the field of tritium and mass spctrometry. One of my main questions is why is never reported a mass of a tritium labelled peptide? Is it actually possible? For C14 I was able to measure the mass, but not 3H... I do consider the high specificity of eg. 3H in comparation with 14C. One more thing, if one calculates 1 mCi of 3H with 60 Ci/mmol gives an amount of 16.6 nmol...for HPLC, of course one takes a small sample so one gets in the pico area... how is able one to still have an UV peak in the HPLC?
Thank you!

Offline OC pro

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Re: mass spectrum of tritium labelled peptides?
« Reply #1 on: November 19, 2012, 11:19:07 AM »
I do radiolabeling of peptides. Of course in the UV you will not see so much only very tiny spots. But if you use a gamma detector you can measure the radiolabelled peptide.

Offline msd

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Re: mass spectrum of tritium labelled peptides?
« Reply #2 on: November 20, 2012, 05:10:37 AM »
yes, you are right! theoretically one should have almost no UV signal, the radiodector signal in the HPLC and the LSC give you the proof. But tha tis not the problem.
So,
I have synthesized a peptide using the SPPS protocol with Fmoc-AA labelled with 3H (2 tritium atoms). I have purified and I have 1 mCi, but incredible high UV signal...dilution series shows me a pure compound, from the amount I have really a lot (I can actually see the substance, that is in the conditions where 1 mCi is  16 nmol! and my peptide is under 2000!!) so I did a mass with the same conditions as the cold peptide and I see a mass higher with 10 amu.
So, my questions: how come I have a signal so high in the UV? and so much amount that I can measure mass....in literature there is NO report of mass of any tritium labelled peptide...or maybe you know one?

Offline Wastrel

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Re: mass spectrum of tritium labelled peptides?
« Reply #3 on: November 20, 2012, 06:06:56 AM »
Do you know how much unlabeled compound is mixed in with your labeled compound?

Offline msd

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Re: mass spectrum of tritium labelled peptides?
« Reply #4 on: November 20, 2012, 06:33:52 AM »
there shouldnt be any unlabelled material because when I couple the labelled AA  (which I have also synthesized) I do make acetylation of the peptide and then proceed further! And anyhow, when that would be the case that is a mixture of unlabelled and labelled material, then I should see it also in the mass! By 2 T there is a difference in 4 amu, so that should be easily seen. Furthermore, when I mix cold and labelled peptide I do see both peaks, corresponding to each peptide, cold and labelled, but with a difference of 10 not 4! 

Offline gippgig

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Re: mass spectrum of tritium labelled peptides?
« Reply #5 on: November 23, 2012, 04:02:30 AM »
A labeled compound generally consists almost entirely of the regular compound with only a trace of the labeled form. Therefore the measured molecular mass won't differ - the amount of the labeled form is simply too small to detect.
I have no idea why you are measuring a difference of 10; that doesn't make sense. Are you sure you have the right compound?

Offline msd

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Re: mass spectrum of tritium labelled peptides?
« Reply #6 on: November 23, 2012, 09:16:34 AM »
you mean when you buy a labeled AA it comes as a mixture of cold and labeled although it has the highest specific activity? I thought that one adds cold material to dilute the specific activity...
well,  meanwhile I have analyzed my original AA[3H] which came from the company and I see a mass 3 units smaller than the cold AA...if there is a mixture from cold and labelled material, it should be the same mass as the cold, isn't it?...
Do you know of any paper where they measure mass of tritium compounds?
thank you for your input!

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