Dear Susana Linskens:
In our experience (1), using an ABI 477A sequencer with their standard
chromatographic system, the PTH derivative of Asn with one GlcNAc residue on
the side chain, Asn(GlcNAc), will elute between DTT and PTH-Asp. Perhaps your
PNGase enzyme was contaminated with some EndoH, as EndoH would cleave between
the core GlcNAc residues of oligomannose structures, leaving the Asn(GlcNAc).
Ray Paxton et al. (2) published a chromatographic method that provides better
resolution for PTH-Asp and PTH-Asp(GlcNAc).
I don't think the derivative would be isoAsp, because isoAsp is not
susceptible to the Edman degradation. The extra -CH2- in the polypeptide
backbone prevents formation of the ATZ, halting the Edman degradation.
The Asn could be due to partial glycosylation at this site.
Best regards, - Reed Harris, Genentech Analytical Chemistry Dept.
1. J. Biol. Chem. 265, 10373-10382 (1990)
2. PNAS USA 84, 920-924 (1987)
Susana Linskens wrote:
> Dear ABRF:
> This is a question for these guys with experience in sequencing
> deglycosilated proteins.I treated a reduced plus 4-VP protein, with PGNase.I
> used the vendor suggested conditions. For " desalt " the mixture, I used a
> short C8 column, eluting with the usual 0.1% TFA-Acetonitrile buffers.At the
> supposed N-glycosilated position, I found a small Asn signal, plus a smaller
> peak, between DTT and Asp ( perhaps Iso-Asp ? ). As far as I know, I would
> wait only Asp there. Any suggestions ?
> Thanks in advance for your input.
> Susana L.
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> Susana Linskens
> LANAIS-PRO
> Junin 956
> 1113 Buenos Aires - ARGENTINA
> linskens@qb.ffyb.uba.ar
> FAX: 54-11-4508-3652
> Phone: 54-11-4508-3651
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