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Dear ABRF,
Our lab would like to quantitate the number of O-linked sugars on
glycoproteins. We would like to do this while consuming 10 ug or less
glycoprotein. I suspect that others would like to do this also. Is anyone
aware of a method for determining the numnber of O-linked sugars on a
glycoprotein that consumes 10 ug (200 pmol) or less material? The methods I
am aware of are not at all sensitive and are summarized below. Can any of
these be modified to improve sensitivity? Anybody with practical experience
they can share?
1. Alkaline borohydride treatment releases oligosaccharides and leaves an
N-acetylaminitol at the former reducing end. Acid hydrolysis produces
monosaccharides which (after re-N-Acetylation) can be separated by high pH
anion exchange chromatography with pulsed amperometric detection. This
technique seems to require 500 ug (10 nmol) or more or more hexosaminitol
for quantitation, probably because of the effects of contaminating salts.
2. One can release sugars in the same way, acid hydrolyze and make PITC,
OPA, or FMOC derivatives of amino sugars and separate using amino acid
analysis chromatography systems with UV or fluorescence detection. A
published method indicates a linear standard curve from 1-70 nmol of
hexosaminitol, although results for hydrolysis of an actual glycoprotein are
unimpressive.
3. One can treat the glycoprotein under mild conditions with
trifluoromethanesulfonic acid, leaving GalNAc still attached to Ser/Thr
residues. Then one sequences peptides by Edman or, I suppose, uses mass
spectrometry. In a 1997 JBC article, authors used 0.75 grams of
glycoprotein with the Edman approach.
Thanks in advance for help with this.
Joe
Joe Zaia
Osiris Therapeutics, Inc
2001 Aliceanna St.
Baltimore, MD 21231-2001
Phone: 410-522-5005 X 297
Fax: 410-522-6999
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Dear ABRF,
Our lab would like to quantitate the = number of O-linked sugars on glycoproteins. We would like to do = this while consuming 10 ug or less glycoprotein. I suspect that = others would like to do this also. Is anyone aware of a method = for determining the numnber of O-linked sugars on a glycoprotein that = consumes 10 ug (200 pmol) or less material? The methods I am = aware of are not at all sensitive and are summarized below. Can = any of these be modified to improve sensitivity? Anybody with = practical experience they can share?
1. Alkaline borohydride treatment = releases oligosaccharides and leaves an N-acetylaminitol at the former = reducing end. Acid hydrolysis produces monosaccharides which = (after re-N-Acetylation) can be separated by high pH anion exchange = chromatography with pulsed amperometric detection. This technique = seems to require 500 ug (10 nmol) or more or more hexosaminitol for = quantitation, probably because of the effects of contaminating = salts.
2. One can release sugars in the same = way, acid hydrolyze and make PITC, OPA, or FMOC derivatives of amino = sugars and separate using amino acid analysis chromatography systems = with UV or fluorescence detection. A published method indicates a = linear standard curve from 1-70 nmol of hexosaminitol, although results = for hydrolysis of an actual glycoprotein are unimpressive.
3. One can treat the glycoprotein = under mild conditions with trifluoromethanesulfonic acid, leaving = GalNAc still attached to Ser/Thr residues. Then one sequences = peptides by Edman or, I suppose, uses mass spectrometry. In a = 1997 JBC article, authors used 0.75 grams of glycoprotein with the = Edman approach.
Thanks in advance for help with = this.
Joe
Joe Zaia
Osiris Therapeutics, =
Inc
2001 Aliceanna St.
Baltimore, MD =
21231-2001
Phone: 410-522-5005 X =
297
Fax: 410-522-6999