Relative quantitation of N and O-linked glycans by isotopic permethylation
using 13CH3I and Mass spectrometry

Purpose:
This technique has been used to quantify and compare the glycans between glycoproteins coming from animal cells subjected to genetic mutations [i.e. wild vs. mutant species; Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42].

Methods used:
The glycans released from one source are permethylated with [12C] methyl iodide, whereas glycans released from another source are permethylated with [13C] methyl iodide.  Following permethylation, the labeled glycans are mixed together on an equal protein basis and analyzed by Mass spectrometry.

General Criteria for the sample:
The amount of the starting material required for N- or O-linked oligosaccharide profiling will depend on the substrate or the number of the glycosylation site.  If a sample is a single purified glycoprotein, 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:

ESI full-MS spectrum of permethylated N-glycans. Above-mentioned technique was used.