amino acid analysis of glycoproteins

Steven Pirie-Shepherd srps at galactose.mc.duke.edu
Tue Apr 30 21:05:16 EST 1996


Ray Oomen (roomen at HOOKUP.NET) wrote:

: 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.

: Ray Oomen
: roomen at hookup.net

--

How about removing the Triton by soem precipitaion technique (TCA?, AS?), 
then doing the 6N HCl. This will certainly destroy the NeuNAc, and may 
well destroy the neutral sugars. I think the only sugars to survive this 
treatment would be amine sugars, and they might casue some strange 
'blips'. Having said that, I have succesfully managed to perform AA 
analysis on glycopeptides, where the CHO is at least 10-20% of the mass 
of the peptide. Or rather someone did the analysis for me, and it agreed 
pretty well with the theoretical numbers. (The peptide was of known 
sequence, sequencing was used to determine which residue was modified 
with CHO, by looking for  a blank cycle in the Edman degradation reaction).

Hope some of this is of some help.

__________________________________________________
Steven Pirie-Shepherd		       		
srps at galactose.mc.duke.edu	        
"Insert your own pithy phrase just about here!"



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