Re: ProtSeq of native disulfide bonds
Gregory Grant (ggrant@pharmdec.wustl.edu)
Thu, 22 Oct 1998 23:34:18 -0500
>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
Robert,
PTH-cystine does indeed elute with PTH-tyrosine. My recollection is
that the yield is variable and decreases with the number of cycles into the
sequence. Check out Burman et al., PNAS 86, 429-433, 1989.
Gregory A. Grant
ggrant@pharmdec.wustl.edu