Dear all,
at first, thanks to all who sent me their hints and comments about the
reduction of Met-O. I finally decided to use N-methylmercaptoacetamide (pH
8, 100 mM NH4HCO3, peptide conc. 5mg/ml and reduction over 72h, well,
actually over the weekend...) and it worked fine in two cases where I
observed Met-O. In the case of the peptide for which I originally asked, it
didn't. In MALDI-MS I observed a modification with a mass shift 75.1 Da,
which is stable to the reduction with DTT (the mass shift is observed only
by one modification at the N-Terminus and the Met-O was properly reduced;
this was checked by MALDI-PSD). The peptide contains an additional cystein
which is the place of modification (I actually don't know wether the
N-terminus or the side chain is modified, but I assume the latter one). Is
anybody of you aware about the nature of this modification? All
possibilities I calculated don't make very much sense (e.g. cleavage of the
amide bond in the acetamide etc.).
Please dont' worry if you can't figure out since this a merely academic
question (we have resynthesized the peptide and cleaved it of with 94% TFA,
2.5% water, 2.5% TES and 1% EDT; it smells awful, but it avoids oxidation...)
Yours sincerely,
Marcus
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Dr. Marcus Macht
Universit”t K–ln
Zentrum f¸r molekulare Medizin
Zentrales Servicelabor
Joseph-Stelzmann-Str. 52
50931 Koeln
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail: Marcus.Macht@uni-koeln.de
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