At 01:30 PM 1/15/99 -0500, Henry Keutmann wrote:
>
> We are studying the effects of methionine oxidation on the
function of
> a protein that also contains tryptophan. Following conversion of Met to the
> sulfoxide by means of hydrogen peroxide (1:50), we find upon sequence
> analysis that tryptophan has been largely replaced by an derivative eluting
> earlier, just before proline, from our ABI 477 system (time 19.03 min,
> compared with Pro 19.93, DPTU 23.33, bona fide tryptophan 24.95 min on the
> usual NaAcetate/ isopropanol/acetonitrile gradient). Tryptophan appears
> normal in control preparations incubated with solvent alone (.1% TFA pH 2.0,
> 37 deg, 45 min).
> Reports I've found, dating back a long time, indicate that tryptophan
> is not affected by these oxidation conditions. Has anyone seen this
> derivative, and if so does it represent serious damage or modification prior
> to sequencing?
I don't know its elution position in the ABI, but I suspect that you have
kyneurine (or, less likely, N-formyl kyneurine) which is a major oxidation
product of tryptophan. It's extremely easy to produce this when proteins are
treated with ozone, but other oxidizing agents can do it as well. If possible,
check the mass of your peptide by MALDI or ES and see if it matches.
Rod Levine
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At 01:30 PM 1/15/99 -0500, Henry Keutmann wrote:
We are studying the effects of methionine oxidation on the function of a protein that also contains tryptophan. Following conversion of Met to the sulfoxide by means of hydrogen peroxide (1:50), we find upon sequence analysis that tryptophan has been largely replaced by an derivative eluting earlier, just before proline, from our ABI 477 system (time 19.03 min, compared with Pro 19.93, DPTU 23.33, bona fide tryptophan 24.95 min on the usual NaAcetate/ isopropanol/acetonitrile gradient). Tryptophan appears normal in control preparations incubated with solvent alone (.1% TFA pH 2.0, 37 deg, 45 min).
Reports I've found, dating back a long time, indicate that tryptophan is not affected by these oxidation conditions. Has anyone seen this derivative, and if so does it represent serious damage or modification prior to sequencing?