I've been sequencing some peptides that came from a protein that had
been reduced using tetrathionate. When we get to the Cys sites (side
chain: -CH2-S-SO3H), we get a huge jump in lag. For example, suppose I
have an -Ala-Cys(SO3H)-Leu- sequence, the data look like this:
cycle N-1 100 pmol PTH-Ala
cycle N blank cycle
cycle N+1 15 pmol PTH-Leu
cycle N+2 40 pmol PTH-Leu
cycle N+3 25 pmol PTH-Leu
The lag is permanent, i.e., the rest of the data are lagged. We see
this with both our ABI and H/P sequencers.
What's going on here? One guess is that the cysteine beta-eliminates,
giving a =CH2 side chain, with the double bond migrating to the
polypeptide bond (thus mostly blocked), with some reversion to the
side-chain double bond (or reduction) during the acid cleavage or
subsequent base incubation, allowing the Edman degradation to proceed
again. Has anyone tried to sequence peptides with serines that have
been deglycosylated by beta-elimation? if so, do they give this same
pattern?
Or, might this be similar to the O ---> N acyl shifts that has been
reported for acetylated serines? (in this case, an S ---> N shift) if
so, why does it unblock?
Thanks in advance...
-- Reed Harris Genentech, Inc. (650) 225-4187 Phone (650) 225-3554 FAX reed@gene.com E-Mail