Glycosylation site mapping

Purpose:
Identifying the site(s) of attachment of N- or O-linked glycans.

Methods used:
Usually, 18O -labeling method are used to elucidate N-linked site(s). Briefly, sample are digested with protease and the glycopeptides are deglycosylated with PNGase A/F in H218O converting the asparagine into an aspartic acid. A mixtrue of pepteids are then analyzed by C18 capillary chromatography LC/MS. The data acquisition process included a full MS scan followed by MS/MS fragmentation of the most intense ion selected from the full MS spectrum. On the other hand, BEMAD (β- elimination followed by Michael addition with dithiothreitol) can be used to identify O-linked sites.

General Criteria for the sample:
A single purified glycoprotein is recommended for this analysis.  The amount of the starting material required for this analysis will depend on the substrate or the peptide sequences.  Approximately, more than 30ug (500pmol) of a glycoprotein will be needed to see reliable result but more material will be needed if a sample is a crude protein.

Example Results:

MS/MS spectra showing localization of (K)NERFWN*TTNPIETTAYALLSFVMAEK(Y)glycosylation site