<x-charset iso-8859-1>Original message by Thomas Weber:
<< H W Y D S F V P W G H Q C
[...]
....when I rerun the sample the sample seems to be actually less pure
than the original crude peptide (which was >90% pure). The additional
peaks I see elute a little later than the original peptide and look
similar to the oxidation products I observed earlier. Could it be that
this is air oxidation ? I kept the peptide as a lyophilized powder
at -20°C over drierite. Any suggestions what might be going on and how
to prevent this degradation ?
...
Thomas>>
Dear Thomas:
Do not worry about "this degradation" that much. This behaviour is
typical of peptides having free HS-groups and HPLC profiles should be
taken with understanding rather than literary. Air oxidation of unstable
thiol function can not be avoided completely, and there are only
invasive approaches to keep the process at minimum. Depending upon
future application of your peptide, you may select one of the following
methods:
i) seal freshly lyophilized peptide in glass under vacuum after thorough
flashing with argon, He or nitrogen;
ii) keep the peptide in presence of DTT, mercaptoethanol, Bu3P, TCEP
either in solution or as a lyophilized powder (preferably with TCEP and
DTT) in inert atmosphere in tightly capped vial;
iii) = ii), but omit reducing stabilizer for short storage periods. Open
in the gentle flow of argon. I recommend to lyophilize and store peptide
directly in a Schlenk vessel (see Aldrich LabGlass pages), which is
equipped with a stopcock allowing to handle substances under isolating
flow of argon etc. Can be always found in any organoelement chemistry
lab.
IMHO, via lyophilizing "a few times in succession", one can actually get
rid of the smell of reducer, but 0.01% of oxidized thiol or phosphine
will persist.
Regards,
Igor Rodionov
Laboratory of Peptide Chemistry
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
8 Academy Avenue, Pushchino, Moscow Region
142290, Russian Federation
rodionov@fibkh.serpukhov.su
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