<< I have a glycoprotein with a highly hydrophobic peptide backbone that
remains
soluable when glycosylated. However, when I digest and deglycosylate,
the hydrophobic peptides precipitate from solution. By holding the solution
in
guanidine, I am able to arrest any visible precipitation however, my
recovery of
2 peptides is only 50%. These peptides have ~30% leucine or isoleucine
residues.
Ugh. The remaining peptide peices are not very hydrophobic and are recovered
well. At present I am using a Jupiter C4 column with water/acetonitrile/tfa
mobile phase.
Are there any ideas about how to increase my recoveries for these 2
peptides? I
am guessing that they're lost in the tube but perhaps they are lost once
they're
injected. Has anyone put guanidine in their mobile phase?
Thanks for your help.
Shawn Novick
****************************************************************************
Shawn - you might consider omitting much or all of the Gd.HCl and letting the
two peptides precipitate quantitatively. Remove the supernatant for regular
C-4 HPLC purification. To the remaining precipitate, add 70-85% propanol
containing 50 mM formic acid (it may also be helpful to include 50 mM
hexafluoro-2-propanol). If that succcessfully dissolves the precipitate,
then you can apply the resulting solution directly to an HPLC column of
PolyHYDROXYETHYL Aspartamide equilibrated with the same solvent, then run a
gradient of decreasing propanol concentration to elute the peptides This is
hydrophilic interaction chromatography (HILIC). Retention will be a function
of whatever polar residues the peptides do have; the column will simply
ignore the hydrophobic residues, any attached acyl- chains, etc (the opposite
selectivity of reversed-phase HPLC). For an example of these conditions
applied to an integral membrane protein purification, see
Jeno et al., Anal. Biochem. 215 (1993) 292.
Andy Alpert
PolyLC Inc.
Columbia, MD
tel: (410) 992-5400