ProtSeq of native disulfide bonds
rlundgard@neurex.com
Thu, 22 Oct 1998 12:30:12 -0700
We have successfully sequenced through intact cystine residues of a small
peptide, however
the results are sometimes variable. Does anyone have experience with the
optimization
of ABI 477A sequencing chemistry (reaction cycles, reagent purity, etc.)
for peptide cystines, in order to determine the disulfide bond arrangement?
We are using the standard 477A sequencing protocol with the
conversion temperature reduced from 65C to 50C to improve the yield of the
cystine
derivative. The sample (25 amino acids, 6 cysteines in 3-disulfide
bridges) load is a few
nmol , and recovery of the released cystine derivative is about 1-10% of
other amino acids.
The PTH-derivative coelutes with tyrosine in the A3/B ABI buffers, between
arg and pro. Has anyone confirmed that this is a PTH-cystine.
(Dehydroalanine apparently does not elute with tyr.) Sometimes the
disulfide bridges seem to be reduced in the first cycle of the sequencing
process. (The ABI cleaving TFA does not contain DTT.)
-Regards
Robert Lundgard
Elan Pharmaceuticals (formerly Neurex)
3760 Haven Ave
Menlo Park, CA 94025
Phone: 650-614-1034
Fax: 650-614-4361
email: rlundgard@neurex.com