Boronate chromatography of glycopeptides

Reed Harris (reed@gene.COM)
Thu, 18 Mar 1999 10:35:44 +18711

ABRF colleagues:

We've had some success resolving glycated and non-glycated forms of
proteins and peptides using the TSK-boronate column. In our experience,
this column does not bind N-linked glycopeptides. The key difference is
that glycopeptides have sugar groups that are their ring conformations,
wheras glycation results in an Amidori product that is in a linear form
with cis diols that bind to the stationary phase.

For an example of this technique, see Felicity Shen's poster
at this weekend's ABRF meeting. I have to miss the ABRF meeting due
to a conflict with a project-related meeting here in SSF.

Best regards,

--
Reed Harris
Genentech, Inc.
(650) 225-4187 Phone
(650) 225-3554 FAX
reed@gene.com  E-Mail




> We are trying to use boronate affinity columns from TosoHaas to
isolate
> glycosylated recombinant proteins and glycosylated peptide fragments
> produced upon proteolytic digestion of the recombinant glycoproteins. 
These
> are primarily, but not exclusively N-linked glycosylation.  As an
> alternative to Lectin Affinity chromatography,  the use of the
boronate
> columns sounds attractive because we could simultaneously isolate N-
and
> O-linked glycopeptides from non-glycosylated peptides and elute the
> fractions in mobile phases that are relatively friendly to subsequent
LC/MS
> and CE/MS analysis.  A suggestion has been made that boronate columns
work
> well for glyco-amino acids, less well for glycopeptides and poorly for
> glycoproteins.  Can you shed some light on this for us.  
> 
> Alex Apffel, Ph.D.
> Biomeasurements Group
> Hewlett-Packard Laboratories
> 3500 Deer Creek Road
> Palo Alto, CA 94304 USA
> Tel 650-857-6090
> Fax 650-852-8502
> email alex_apffel@hpl.hp.com 

--
Reed Harris
Genentech, Inc.
(650) 225-4187 Phone
(650) 225-3554 FAX
reed@gene.com  E-Mail