At 16:44 20.11.00 -0800, you wrote:
> Could someone please tell me how glycosylation effects protein
>sequencing? THANKS, Linda Linda De Young, Ph.D.
>Vice President, Product Development
>Angiogenix, Inc.
>875-B1 Cowan Road
> 94010
>
Dear Linda,
after David Chiara's comments on N-glycosylation, I want to add my two
cents on
O-glycosylation. We have sequenced a couple of O-glycosylated peptides on
our ABI473, which were carrying GalNAc residues at Ser as well as Thr. The
sequencing reaction works fine even if the residues are glycosylated. What
you will get in the chromatogram afterwards is a splitted peak due to
different diastereomers. HexNAc-Thr will look like Ser and Gly, HexNAc-Ser
will show a more or less seperated doublet near the Asp peak. Due to
incomplete glycosylation we mostly observed the unmodified residue as well.
One major problem is incomplete cleavage since O-glycosylation often
occures in Thr or Ser clusters, which will make quantification difficult.
The reason for this is that you cannot distinguish between an only
partially modified residue and a mixture between e.g. fully modified
residue and remainings of unmodified residue from the previous.
Summarizing it up, sequencing of O-glycosylated residues works fine with
standard conditions but quantification is difficult.
Yours sincerely,
Marcus
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Dr. Marcus Macht
Universit”t K–ln
Zentrum f¸r molekulare Medizin
Zentrales Servicelabor
Joseph-Stelzmann-Str. 52
50931 Koeln
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail: Marcus.Macht@uni-koeln.de
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