>>In the current CNBr digestion discussion, Deb McMillen mentioned the
>>problem of recovering large CNBR generated peptides by RP-HPLC. Now th=
at
>>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 compara=
ble
>>sized "standards", such as insulin and myoglobin, presumably because th=
ey
>>have association properties that are required for assembly of the prote=
in
>>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 re=
sins
>>and buffers they have used.
>>
>>We also have problems seeing these large CNBr or trp cleavage peptides =
out
>>of MALDI matrices (we are new to maldi and use the prepared matrices fr=
om
>>HP). I would also be interested in any hints about alternative matrice=
s
>>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 p=
H
>>5.0 in NH4OAc to 15% HOAc. The large fragments will have more charge t=
han
>>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 oper=
ate
>>in that mode too.
>>
>>HIC is a poorer choice since the solvating capability goes away with th=
e
>>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 agai=
nst
>>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 too=
ls,
>>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 ta=
lk
>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 =
you
>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 custome=
r.