Re: MS/MS of peptides with disulfide bridges
Katheryn Resing (uunet!Colorado.EDU!Katheryn.Resing@mail.uu.net)
Wed, 4 Mar 1998 08:38:02 +0000
Hi, I've done a lot of MS/MS on disulfide bonded peptides. A 15-20 mer
with 2 disulfide bonds will probably be fairly difficult because residues
near the "loops" probably won't open. I assume they want to assess the
extent of heterogeneity in the bonding. I used two techniques to assess a
similar problem, using a sciex api3+ (with a high pressure collision cell),
I got reasonable fragmentation at the orifice that took it down to the
"core" part, which then fragmented sufficiently for me to diagnose it.
When combined with the fact that the various forms resolved on a high
carbon loaded C18 column (Vydak often works), we could sort it out.
Fragmentation at the CC dissulfide produces a series of ions that can be
32-34 Da different from the cys mass. It helps to do a careful study of
the fragmentation of the reduced peptide as well (not reduced and
alkylated, just reduced).
The other approach will work only if you can resolve the various forms and
get them into separate tubes. In the looped peptides I did, I used
digestion with pepsin to cleave the peptide (pepsin is nice because its
done in acid, so there is less rearrangement of the disulfides). This
produced opening of the "loops" (the number of openings can be counted by
looking at the number of waters that add to the mass of the peptide), but
they were still hooked together. MS/MS of this peptide then gave
information about how the pieces were hooked together. It occurred to me
at the time, that partial acid digestion might be useful in this type of
application, but I never tested it.
Katheryn Resing