I come into this after many interesting replies.I have another
outlook,since I have quit doing any sequencing long ago.
However,with the advent of AccQTag chemistry,I have been using acrylamide
as alkylatator of cysteine after hydrolysis.Acrylamide reacts quite
rapidly,as pointed out elsewhere,so it competes with the quinoline reagent
during the derivatization,and,there is minimal dimer formation under basic
conditions,even 60.I prefer to alkylate after hydrolysis,because the S-Pam
Cysteine elutes in a discreet peak,between Ala and Pro,while the more
acidic deaminated compound would coelute with Ala.So,there is ample
evidence that there can be alkylation of free Cys.
I would expect that a certain amount of the deaminated derivatives would
be present.There is a paper by D.C.Brune in Anal.Biochem.207
,285-290.1992,which discuss the alkylation of cysteine for sequence
analysis.According to this paper PTH-Cys-PAM elutes between Glu and
His,while PTH-Cys-S-propionic elutes near Ala,in his system.Are we talking
about the same derivative,I would not know,because of different elution
conditions.
Fulvio Perini
UNMC
986805 Nebraska Med.Cntr.
Omaha,NE 68198-6805
Sandy Kielland
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04/26/2001 06:29 PM
Hello all;
I recently performed a sequence analysis on a blotted protein and obtained
a residue with a non standard retention time almost identical with that of
DMPTU. A database search on the first fifteen residues found a perfect
match with a cysteine in the position in question. I found out from the
researcher that the sample had not been alkylated prior to isolation. The
acrylamide derivative of cysteine (PTH-Cys-S-Pam) elutes in this region and
it seemed reasonable to assume that it had been modified because the gel
was not polymerized long enough. However, he has repeated the procedure
three more times with ample polymerization time, but I still see the same
results. Has anyone out there had a similar experience?
Also, if there were enough free acrylamide around to completely derivatize
the cysteine (the peak was the same height as the other residues),
shouldn't it have blocked the N-terminal of the protein as well? I got an
excellent initial yield (~20 pmole) from a single minigel band.
Thanks in advance for any input...
Sandy
Sandy Kielland
Victoria Protein Microchemistry Centre
Department of Biochemistry and Microbiology
University of Victoria
Victoria B.C. CANADA
phone: (250) 7218884
FAX (250) 7218855
Email Kielland@UVic.ca
Webpage http://www.proteincentre.com
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