RE: acrylamide cys
Joseph Fernandez (fernaj@rockvax.rockefeller.edu)
Thu, 26 Feb 1998 07:47:54 -0500 (EST)
Steve,
I agree with you about acrylamide being a typical side reaction
(not sure of one third though). I disagree with the statement that protein
derivatized with two different alkylating reagents will cause a shift in
retention time. We have been digesting SDS-PAGE purified proteins with
reduction and carboxyamidomethylation for 3 years. We have observed many
examples of the same peptide with both CAMC and acrylamide cys (PAMC)
eluting in the same HPLC peak, proven unambiguously using MALDI-TOF MS and
Edman Sequencing. We actually use this as a tool for screening HPLC
fractions for Edman sequencing as the difference in mass between the two is
14 daltons. When we observe a mass spectra with two masses, 14 daltons
apart we have a good chance to get a cysteine in it. We have actuall been
able to get a few peptides with multiple cys's by observing +14, +28, and
+42.
It's similar how we try and predict MET and TRP's using MS by
looking for +16, and +32. However in this case the peptides containing
oxidized MET's and TRP's will elute earlier on HPLC, and the oxidized MET
we observe in the MS profile represents probable oxidation within the
instrument. We usually try to confirm MET containing peptides by also
looking for an earlier eluting peptide that is 16 ot 32 daltons higher.
CYS, MET and TRP or preferred residues to sequence through as they
tend to be conserved and provide nice primers for thse investigators who
still work with uncharacterized proteins. Yes there are still quite a few
out there that are not in the databases.
Joe
Joseph Fernandez
Associate Director
The Rockefeller university
Protein/DNA Technology Center
1230 York Ave. New York, NY 10021
Phone: (212)-327-8869
FAX : (212)-327-8620
email: fernaj@rockvax.rockefeller.edu
Lab Web Page: http:\\pdtc.rockefeller.edu