RE: membrane spanned peptides

Amina (amina@welchlink.welch.jhu.edu)
Thu, 14 May 1998 09:28:26 -0400

This is the type of problems where using a polyethylene membrane comes in handy! Obtain a polyethylene membrane from 3M, activate the membrane with 1-2 ul methanol, immediately deposit your peptide mixture, let it dry at room temp, wash with 100-200ul 70% methanol, let membrane dry, deposit 1 ul matrix solution (saturated a-cyano-4-hydroxycinnamic acid in 50% ethanol), let it dry. Affix the membrane to a MALDI target, and acquire a spectrum. This should be enough. If you need more details read Worrall et al. Anal. Chem. 1998, 70, 750-756.

Amina

Amina S. Woods, Ph.D.
Johns Hopkins School of Medicine
725 N Wolfe St., Baltimore, MD 21205
Tel: (410) 614-4981, Fax: (410) 955-3420
E-mail: amina@welchlink.welch.jhu.edu

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From: Suming Hua [SMTP:shua@umaryland.edu]
Sent: Wednesday, May 13, 1998 3:31 PM
To: Recipients of ABRF List
Subject: membrane spanned peptides

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.