ABRF members,
Two points regarding Edman. My opinion is that Edman has not been
forgotten but more accurately is not the main focal point of a protein
characterization lab, obvious MS is and should be. The second point is
that Edman produces primary sequence information in a completely different
way from MS/MS sequencing. Seams best to have 2 different approaches at
one's hands to unravel scientific problems and not rely solely on
one. There are plenty of instances where MS/MS will not work (if I am
wrong then please someone let me know if they always get MS/MS data) just
as there are many instances where Edman clearly falls short of what MS/MS
can provide.
Some points about modified cysteine
1) The derivative observed is definitely acrylamide modified cysteine. As
someone pointed out you may also be able to see the propionic acid
breakdown peak eluting just before ALA.
2) acrylamide modification of the N-terminus is extremely slow and is
pretty insignificant. We did a series of Edman studies several years ago
for intact protiens and peptide fragments and saw no significant
differences in initial yields.
3) I have observed the self casted gels are much more likely to generate
acrylamide-modified cysteine than most commercially prepared precast gels.
4) I disagree that acrylamide is the best derivatization reagent. You
usually require 1 M solution to get complete modification (2 hrs at 55C)
which is rather harsh to put on a reverse-phase HPLC. I have also observed
by MAldi-Tof MS acrylamide aducts of +71 in addition to the modified
cysteine (sometimes up to 5 additions).
5) The classic iodoacetamide and iodoacetic acid derivatives are my
personal favorites. Providing complete reaction in a limited time at room
temperature and not interfering with HPLC or MS (providing you use a ziptip).
6) Finally, I have rarely found the acrylamide derivitization on a gel to
be complete. This determined by treating the gel band with iodoacetamide
before digestion and observing a CAMC and PAMC derivative both by MS and Edman.
Joe
Joseph Fernandez
Associate Director
Protein/DNA Technology Center
The Rockefeller University
1230 York Ave.
NY,NY 10021
Phone: 212-327-8869
FAX: 212-327-8620
Email: fernaj@rockefeller.edu
website: pdtc.rockefeller.edu
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