I have had no problem dissolving peptides in neat formic acid.The peptides
are dissolved just before injection.But you
do not know how soluble they will be after the formic is gone.I have seen
peptides which were difficult to dissolve in
any of the common solutions,but after purification they were ok.So,you will
have to be careful.Another approach,which
is used by one of my colleagues,is to use triethylammonium phosphate,at
whatever pH you choose(he claims that he
learned the procedure from someone at Scripps).He uses relatively high
concentration of acetonitrile,but since B is
only 60% acetonitrile,you need less organic to elute the peptide.He
lyophilizes the eluent,and gets an oily mess,more
often than not..All the salts will be there.He then reruns the
sample,replacing the A buffer,with one containing acetic acid.
You can easily see where the TEAP is replaced by the acetic acid,if you
monitor for the carbonyl group at about 220 nm.
Whichever method you use,will probably require a pH of 5 or higher,and we
will have to start with semi-prep level samples to prevent heavy losses.
lisa bibbs <bibbs@scripps.edu>@aecom.yu.edu> on 08/30/2000 06:14:28 PM
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cc: peptide@scripps.edu, woodside@scripps.edu
Subject: PepSyn:solubilizing and purifying phospho peps
Hello ABRF peptide guru's!!
I have three peptides that have two phospho Y's each.
One has overall -5 charge(19mer), -3 (19mer), and -2 charge (23mer).
All precipitated during cleavage in their TFA. I have been able to
get the one with -3 charge in solution at about 0.5 mg/mL slightly
acidic water. The others are more like 0.1 and I have 0.25mMol Synth
to purify.
The HPLC traces look like a forest but the Mass specs are pretty
clean. I got the -3charge purified on a c18 but when I run
analyticals of the purified material, it reverts to multiple peaks
again. I checked by MS of the various fractions I got from the prep
and low and behold they have the same mass so I have multiple charge
species that run at different retention times.
I've thought about dissolving the other two in Guanidinium HCl but I
am worried about them ppt'ing during purification on my column let
alone the fact that I will probably get multiple species again
although their analyticals look much better.
Any purification suggestions would be appreciated.
Thanks,
Lisa
Lisa Bibbs, Ph.D.
10550 N. Torrey Pines Rd.
SP3
La Jolla, CA 92039
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