Re: MS and HPLC:lg peptides

Deb McMillen (mcmillen@morel.uoregon.edu)
Fri, 20 Feb 1998 13:07:54 -0800

>In the current CNBr digestion discussion, Deb McMillen mentioned the
>problem of recovering large CNBR generated peptides by RP-HPLC. Now tha=
t
>mass spectrometers can more effectively analyze large peptides, we are
>starting to generate larger fragments, using CNBR or tryp cleavage. We
>also have run into this problem of desalting/purifying larger peptides.
>These digested peptides seem to be much more difficult than the comparab=
le
>sized "standards", such as insulin and myoglobin, presumably because the=
y
>have association properties that are required for assembly of the protei=
n
>structure.
>
>I would appreciate it if anyone who has experience purifying or running
>lc/ms on large fragments from CNBr or tryp cleavage would share what res=
ins
>and buffers they have used.
>
>We also have problems seeing these large CNBr or trp cleavage peptides o=
ut
>of MALDI matrices (we are new to maldi and use the prepared matrices fro=
m
>HP). I would also be interested in any hints about alternative matrices
>that have worked with peptides generated by CNBr or tryp cleavage.
>
>Thank you in advance.
>
>Katheryn Resing

Katheryn,

I had a discussion with Amos Heckendorf at the Nest Group about columns
that would work for very hydrophobic peptides. I'm inserting his advice
here.

>
>Try the PolyAspartic Acid (PolyCAT A) column (p/n P1850 204, $485.00),
>rather than the SULFOETHYL, with 30-40% PrOH (isocratic) and run from pH
>5.0 in NH4OAc to 15% HOAc. The large fragments will have more charge th=
an
>the smaller ones so they will elute later. The PrOH will keep it in
>solution and the hydrophilic surface will not bind it as much as even a =
HIC
>column would.
>
>HILIC is more of a gamble to get retention, but the PolyCAT A will opera=
te
>in that mode too.
>
>HIC is a poorer choice since the solvating capability goes away with the
>high water content, and the use of very little amounts of organic cause
>elution of most things, but probably not yours since it won't be in
>solution. RPC has more organic phase but the RPC coating will work again=
st
>you in the recovery after purification. You could use PrOH or HFIP for
>solvating, or BFBA as an ion pair for more hydrophobic peptides.
>
>That is all for now.
>Amos
>
>Amos Heckendorf (nestgrp@world.std.com)
>The Nest Group, Inc., Value Added Resellers of HPLC Columns (Vydac=81,
>PolyLC=81, Hydrocell=81, BioChrom Labs=81, Jordi-Gel=81, Nucleosil=81),
>Electrophoresis gels and Destaining pads, Sialomed=81 MicroDialysis tool=
s,
>and DNA Isolation Kits
>Tel: 800-347-6378, 508-481-6223; FAX 508-485-5736
>HPLC Applications or Molbiol. Protocols at:
>HTTP://world.std.com/~nestgrp/protocols/protocol.html

In addition, I was looking at a Vydac flier (Winter 98 Vydac Advances)
about separation of difficult (i.e. hydrophobic) peptides. You might tal=
k
to them about their Vydac 219TP54 phenyl reversed-phase column--they run
gradients with 5% n-propanol/0,5% HOAc up to 85% n-propanol/0.5% HOAc.
They can be reached at experts@vydac.com or 1-800-542-8421.

I haven't had an opportunity to try either of these out--let me know if y=
ou
do and they work for you.

Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR

No affiliation with either the Nest Group or Vydac--just a happy customer.