Like many of you we are trying to map protein phosphorylation sites by
mass spectrometry. Everybody in the field knows how tricky this can be and
I wanted to find out if there are any tricks on the mass spec end of the
analysis that one can use to get to these sites. Since the stoichiometry of
phosphorylation is often low one encounters the scenario where there is a
relatively high amount of unphosphorylated peptides next to a small amount
of phosphopeptides. This leads to ion suppression and weak signals for the
phosphopeptides. One possible solution to this problem is enrichment of
phosphopeptides by metal chelating resins, a method that apparently doesn't
always work that well due to phosphopeptide loss during the procedure.
Also, the fractionation of peptides during LC/MS/MS can help in finding the
phosphopeptides.
Although the above methods can improve the chances of finding the
phosphopeptides I think there is still a good chance that one will miss
certain sites. The question I have is if one can optimize the mass spec in
a way that favors the ionization and detection of low amounts of
phosphopeptides in a more or less complex mixture with other more abundant
non-phosphopeptides? How about using different buffer systems,
positive/negative ion mode, ionization voltages, etc. to selectively
increase the signal for the phosphopeptides over non-phosphopeptides?
Chris (turck@itsa.ucsf.edu)
This archive was generated by hypermail 2b29 : Fri Jan 05 2001 - 08:42:46 EST