Re: membrane spanned peptides

John Stewart (John.Stewart@UCHSC.edu)
Wed, 13 May 1998 18:57:34 -0600 (MDT)

There are several possibilities to remove hydrophovic peptides stuck to
reverse-phase HPLC columns. One is to do a gradient to 100% acetic acid
(Watch the pressure!!! it will become very high. Slow down the flow).
Another is to do a similar procedure with DMF. Of course, you cannot
monitor eluted peptides using UV detection in these solvents, but you ;may
be able to get it off and detect it in another way (TLC?)
For future reverse-phase work, try a C4 column. Or avoid
reverse-phase altogether. Maybe just silica, with an organic solvent
gradient to elute.

John M. Stewart, Department of Biochemistry
Univ. of Colorado Medical School, Denver, CO 80262
Phone: 303-315-7534; FAX: 303-315-8215
Email: John.Stewart@UCHSC.edu

On Wed, 13 May 1998, Suming Hua wrote:

> I have a problem regarding separation and purification of membrane spanned
> peptides with RP-HPLC. I labeled calcium ATPase on the membrane domain,
> then removed the cytosolic parts of the enzyme with trypsin digestion, and
> pelleted the membrane parts which was dissolved in formic acid, then
> filtered. The labeled peptide was separated and collected with c-18 column
> using TFA-acetonitrile system. When I did further purification, the
> peptide was stuck in the column and nothing came off at all. I don't know
> if there are any other methods to deal with the membrane peptides using
> HPLC system.
>
> How can I identify the labeled peptide in the mixture with mass
> spetrometry (the molecular weight of this peptide is 7-8K)? Is it possible
> to load the mixture on mass spectrometer directly? Thank you in advance
> for your kind advices.
>
>