Re: membrane spanned peptides
POLYLC (POLYLC@aol.com)
Wed, 13 May 1998 21:08:35 EDT
Following tryptic cleavage of the extramembrane domains, solubilize the
subsequent pellet with 85% propanol containing 50 mM formic acid. If
necessary, include 50 mM hexafluoro-2-propanol as well. Apply this to a
suitable column for Hydrophilic Interaction Chromatography (HILIC)
equilibrated with a solvent containing 70% propanol and 50 mM formic acid.
Detergents and salts will elute in or near the void volume. After several
minutes, run a linear gradient to 50 mM formic acid containing 10% propanol.
Under these conditions, proteins and peptides elute in order of increasing
polarity, a normal phase variation. This method works well for membrane
proteins and other proteins not normally found free in an aqueous environment.
The column simply ignores the hydrophobic sections of the peptide and
interacts with the polar residues; surely your peptide has at least a couple
of them. References:
1) Alpert, J. Chromatogr. 499 (1990) 177-196 (introduction to HILIC).
2) Jenoe et al., Anal. Biochem. 215 (1993) 292-298 (removal of SDS and
salts from an electroeluted membrane protein; used a PolyHYDROXYETHYL
Aspartamide column).
3) Swiderek et al., ABRF News 8 (Dec. 1997) 17-25 (HPLC of samples
containing detergents; the membrane protein from ref. 2 is shown in Fig. 4).
Call me if you have any questions.
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: 410-992-5400 e-mail: PolyLC@aol.com