thanks for all the input I got concerning my question as to how infusing a
peptide digest directly into the MS. Just a quick summary, and a couple of
comments to the answers:
The bottom line of the answers was: use stationary phase with higher
carbon loads to maximize binding of hydrophilic peptides. This point is
very well taken. I am a firm believer that the best way to introduce a
sample to a ESI source is chromatography and that there is still hope that
this is doable with short hydrophilic peptides. Just one detail to our set
up: stationary phase is MONITOR C18 material, 100 micron i.d., solvent is
0.5% acetic acid/0.02% HFBA. With this set up we do see the
unphosphorylated peptide which elutes JUST at the start of the gradient.
We assume heavy phosphorylation of this piece, making it so hydrophilic
that under the conditions we are using the phosphopeptides are not
retained on the column. We'll look into different supports.
Of course, Andy Alpert was suggesting hydrophilic interaction
chromatography for these peptides. Good suggestion as well, we have to
look into this.
Ken Mitchelhill suggested using different proteases. As it happens with
phosphorylation sites, the next cleavage site we could exploit produces an
extremely long peptide which is then totally resistant to fragmentation in
the MS.
Thanks again for the input. I'll keep you posted on our progress in
exploiting different stationary phases, since I think this is a frequently
occuring problem.
Paul
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Paul Jenoe, PhD
Department of Biochemistry
Biozentrum of the University of Basel
Klingelbergstrasse 70
CH-4056 Basel
Switzerland
Tel. ++41 61 267 21 68
Fax. ++41 61 267 21 48
e-mail jenoe@ubaclu.unibas.ch
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