Glycosyl-linkage Analysis of Recombinant and Native Glycoproteins

Purpose:
Determination of the linkage positions of sugars in oligosaccharide(s).

Methods used:
Exoglycosidase digestion can be used to determine the glycosyl-linkage with a small amount of sample. First, glycans are released from a glycoprotein sample by enzymatic or chemical cleavage after denaturation and protease digestion. Then the glycans are treated with specific exoglycosidase enzymes such as alpha 1,6 mannosidase, N-acetylhexosaminidase and neuraminidase etc. The linkage is characterized by mass spectrometry. The linkages can also be determined by Partially Methylated Alditol Acetate (PMAA) analysis by gas chromatography-mass spectrometry (GC-MS) but this requires a greater amount of sample.

General Criteria for the sample:
The amount of the material required for linkage analysis by exoglycosidase digestion depends on the substrate or the number of the linkage which you want to identify.  If a sample is a single purified glycoprotein, approximately more than 120ug (2nmol) will be needed to see reliable result but more material will be needed if it is a crude protein. Approximately 4 mg of glycoprotein should be submitted for PMAA analysis by GC-MS.  

MALDI-MS spectrum of permethylated N-linked carbohydrates before (upper figure) and after
 N-acetylhexosaminidase digestion