I tried everything and the only procedure that worked reliably was: After the digest was done I raised the pH of the digest to between 5.0 to 7.0 by adding Ammonium bicarbonate. I'm sure you could use NaOH or any solution that will neutralize the digest. Then I used a RP C4 or C18 4.6 mm X25 cm Vydak columns depending on the resulting protein or peptide fragments.
Amina
Amina S. Woods, Ph.D.
Johns Hopkins School of Medicine
725 N Wolfe St., Baltimore, MD 21205
Tel: (410) 614-4981, Fax: (410) 955-3420
E-mail: amina@welchlink.welch.jhu.edu
Subject: Re: MS and HPLC:lg peptides
>In the current CNBr digestion discussion, Deb McMillen mentioned the
>problem of recovering large CNBR generated peptides by RP-HPLC. Now that
>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 comparable
>sized "standards", such as insulin and myoglobin, presumably because they
>have association properties that are required for assembly of the protein
>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 resins
>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 from
>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