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From: "Paul O'Donnell" <pao@unimelb.edu.au>
Date: Mon, 02 Apr 2001 15:43:01 -0700
Subject: 48 Dalton increase
Organization: The University of Melbourne	

Hi All,
    A peptide is showing a 48 Dalton increase from the MALDI-TOF spectra
(Voyager DE-STR).  There is one cysteine residue in the sequence and was
wondering if the sulphur could hold 3 oxygens. The peptide was generated
via a CNBR digest of a ribosomal protein then HPLC purified. Matrix
alpha-cyano...  Other peptides showed accurate masses (including the
methylated N-terminal peptide)  but none contained cysteine. Not able to
achieve accurate mass of full length protein, approx. 17.2KDa, as it
will only fly in linear mode and the mass varies by up to 65Da  Any
comments on this or direction to sulphur chemistry  references would be
much appreciated.
    Cheers  Paul



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From: l.c.packman@bioc.cam.ac.uk (Len Packman)
Date: Wed, 04 Apr 2001 08:31:46 +0100
Subject: Re: 48 Dalton increase
Organization: Cambridge University

Hi Paul

I've occasionally seen problems with alphacyano matrix giving apparent
oxidation products which were eliminated when doing the same analysis in
sinapinic acid, This is very apparent when Trp is present, which I guess
you do not have in your peptide? If you have then Trp can oxidise for real
and CNBr gives a variety of products.

Len





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From: alex.buko@abbott.com
Date: Sun, 8 Apr 2001 17:18:37 -0500
Subject: Re: 48 Dalton increase
Organization: *


Paul wrote on 4/2/01:
}
}Hi All,
}    A peptide is showing a 48 Dalton increase from the MALDI-TOF spectra
}(Voyager DE-STR).  There is one cysteine residue in the sequence and was
}wondering if the sulphur could hold 3 oxygens. The peptide was generated
}via a CNBR digest of a ribosomal protein then HPLC purified. Matrix
}alpha-cyano...  Other peptides showed accurate masses (including the
}methylated N-terminal peptide)  but none contained cysteine. Not able to
}achieve accurate mass of full length protein, approx. 17.2KDa, as it
}will only fly in linear mode and the mass varies by up to 65Da  Any
}comments on this or direction to sulphur chemistry  references would be
}much appreciated.
}    Cheers  Paul

Cysteine (SH)  is easily oxidized to cysteic acid (SO3H) under appropriate
oxidizing conditions. This would add 48 mass units to your peptide. Typically
performic acid is used to oxidize cysteine to cysteic acid  (Hirs, C.H.W.
(1967) Performic acid oxidation. Methods Enzymol. 11, 59-62 ).  Most amino
acid analyzers can see cysteic acid so that might one approach to confirm. I
don't know that cysteine oxidation is common during a CNBR digest when using
70% formic acid.  Normally cystines are not effected. You may want to
re-evaluate your reagents. The formic acid should be fresh. Typically fresh
ultrapure cyanogen bromide is also critical to limit autoxidation and
formylation of the N-terminus. A small amount of tryptophan (5 mol/mol of
cyteine) may be added to prevent autoxidation.

Alex Buko
Abbott Labs