In article <96Apr30.094932edt.36866 at thebe.connaught.com> roomen at HOOKUP.NET (Ray Oomen) writes:
>From: roomen at HOOKUP.NET (Ray Oomen)
>Subject: amino acid analysis of glycoproteins
>Date: 30 Apr 1996 06:49:44 -0700
>OK, glycochemists, here is a thought experiment:
>Given a small amount of glycoprotein, heavily (and heterogeneously) O-
>and N-glycosylated, and having been purified in the presence of
>detergent (e.g. TX-100), what is the best way to get amino acid
>composition and concentration ?
>Is there anything wrong with traditional hydrolysis with 6N HCl followed
>by HPLC detection of derivatized standards? What would be a suitable and
>sensitive label? Will detergent interfere ? Will the oligosaccharides
>refuse in some instances to come off? I don't think so, but maybe
>someone has actual experience doing this.
>roomen at hookup.net
If you want to remove the detergent for aa sequencing a polishing run down a
reverse-phase hplc column will clean it up and leave it in salt free
conditions. As for the carbohydrate, if it does interfere you can use triflic
acid, (TMS) to selectively remove CH2O and leave the peptide backbone intact.
Oxford Glycosystems do an over-priced kit for this or you could go back to the
papers to get the original method. A combination of both will give you clean
peptide to work with.