Re[2]: Digestion of Toxin, Glu-C at low pH
GARY.W.LANGE@monsanto.com
04 Mar 1999 09:09:35 -0600
Katheryn, I was quite interested in your suggestion that Glu-C (V-8)
would work at a low pH. Even though this has been reported in the
literature (before MS was routinely used for proteins) I have been
totally and absolutely unable to show any cleavage on a protein of any
sort at a pH of 4.0 (the purported lower pH optimum). After trying it
on some half dozen proteins (and I think even a few peptides) I gave
up trying to use it at a low pH. Have you, or any of the other
members successfully used this enzyme at a low pH and obtained greater
than a 5-10% cleavage rate? Don't forget, Asp/Pro cleavages don't
count as they were most likely induced by the low pH. Maybe if a lot
of members respond positively I'll put Glu-C back in the arsenal for
low pH digestions!
I'm assuming that you need to find the disulfide pairs, otherwise you
would just reduce and alkylate. I guess for a tightly knotted
(protein/peptide) that doesn't respond to trypsin, I would try
something like subtilisn or thermolysin, both quite non-specific.
Lots of time points, of course the easiest way to look at this is with
a mass spec. Another useful way (probably the best choice) would be
to try pepsin (at pH 2.0, very nonspecific) again looking at lots of
time points and a relatively low amount of enzyme(1:100 or 1:150). If
you obtain 3 or 4 useful cleavages (as shown by MS) you could then
raise the pH back to 7.5 and try trypsin again. The sites that were
unavailable originally might now be exposed. When trying to obtain a
disulfide bonding pattern one should be a little cautious about
raising the pH too high, especially if incubation is at 37 C.
Depending on the peptide you can cause reshuffling of the disulfide
pairs. (My pH cutoff for a 37 C digestion is 7.5. Above that I have
seen reshuffling. YRMV). Once you obtain some new masses you can
then follow up (using a small aliquot) by reducing the disulfides and
rerunning on mass spec. For MALDI work I use a 10X stock of 20 mM
TCEP (freshly made in water). Incubation with 2 mM TCEP breaks most
bonds in peptides within 30 minutes, incubating a pH 7 or higher at 37
degrees. HPLC separation of linked peptides, followed by TCEP might
make the puzzle easier to interpret.
If you try Glu-C (V-8) at a high pH (7.5-7.8) and have success don't
forget to follow up with a bit of Asp-N. I have found that Glu-C (at
high pH's) often doesn't cleave at aspartic acids. Asp-N works fairly
well, especially as a secondary enzyme when the peptide/protein has
been partially digested. Good luck, sounds like fun!
Gary Lange
Monsanto Co.
gary.w.lange@monsanto.com
ph 314- 737-6602
fax 314-737-7005
______________________________ Reply Separator _________________________________
Subject: Re: Digestion of Toxin
Author: abrf-request@aecom.yu.edu at INTERNET
Date: 03/03/1999 3:06 PM
Your protein is probably also small, am I correct? You probably won't get
it to digest with trypsin. If its fairly acidic, you might have luck with
V8. If you digest it with CNBr, you will probably get large peptides that
may hang together because of all the disulfides. You can get some idea of
whether this will happen by looking at the distribution of cys in the
peptides you expect to be produced.
Why do you want to digest it in the disulfide bonded form? Usually people
want to do this to identify the disulfides. If you do that at neutral pH,
there is a danger that you will get "shuffling" of the disulfides. Thus
for that purpose, one normally digests with pepsin under acid conditions.
Even fairly tightly "knotted" proteins will usually digest with pepsin.
Katheryn Resing