> An investigator has asked me the following:
>
> >Hi Lisa,
> > I need some technical advice. We have three heavily glycosylated
> >proteins which we are planning to analyze by mass spectroscopy, but I'm
> >told that I need to deglycosylate them completely in order to get good
> >results. What do you recommend using? I'm not sure what the full extent of
> >the sugar chains consist of on these proteins, but I know that there are
> >many N-liked moieties at the very least. Is there a glycosidase mix or pool
> >that we could purchase which would cover all the possibilities, and which
> >is pure enough that we could then study the proteins by mass spectroscopy?
>
>
> I don't do deglycosylations so I was hoping someone who does can give me a
> hand.
>
Dear Lisa,
I believe one can get useful information by combining partial
or complete deglycosylation with peptide mapping and MS analysis
methodologies, but need additional information. Are the expected
polypeptide sequences known from genetic techniques or would these
experiments entail formulating de-novo proofs of structure for the
molecules under investigation? Are the three molecules expected to
contain significant stretches of identical or very similar protein
sequence? Can they be separated by electrophoretic or other means? Is
there an analytical method (AAA recommended) by which the
quantity of protein can be estimated? What scale of
analysis are we projecting here - femtomoles,or picomoles, and if the
latter will it be tens of picomoles or hundreds of picomoles? What
quantity of time can be allocated to the work? What
kind of MS analysis is proposed, MALDI-TOF, LC-ESI, other?
Best regards,
Louise
Louise Garone, Ph.D.
Senior Principal Investigator
Ares Advanced Technology
27 Pacella Park Drive
Randolph, MA 02368 U.S.A.
email: louise.garone,us_bos03@serono.com