Purification and analysis of N- or O-linked glycans

Purpose:
Elucidation of the structure of N- or O-glycans released from a glycoprotein.

Methods used:
Glycoproteins are first denatured to make the linkages susceptible to enzymatic digestion.  The glycoprotein sample is then digested with trypsin into peptides.  After trypsin digestion, the sample is treated with N-glycanase such as PNGaseF/A to release the N-linked glycans.  N-linked glycans, and O-glycopeptides and peptides are separated sequentially by passing the sample through a C18 reversed phase cartridge.  O-glycans are released from the peptides by β- elimination procedures, desalted and cleaned of borate.  Hydrazinolysis is sometimes used instead of above-mentioned method (β- elimination).  Whenever necessary, purification steps are undertaken to prepare the N- or O-glycans for permethylation.

Released N- or O glycan fractions can be characterized by the following techniques:

1) Mass spectrometry
Released N- or O-glycans are permethylated and then analyzed by mass spectrometry (MALDI/TOF-MS, ESI-MSn).

2) HPAEC oligosaccharide mapping
Oligosaccharides can be separated by high-pH anion exchange chromatography (HPAEC).  Each peak is collected, desalted, permethylated and then analyzed by mass spectrometry.

General Criteria for the sample:
The amount of the 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) will be needed to see reliable result but more material will be needed if it is a crude protein.  The N- or O-glycans can be prepared not only from a single purified glycoprotein but also cells, tissues, culture supernatants, and SDS gel pieces.

Example Results:

MALDI/TOF-MS spectrum of permethylated N-glycans released from a glycoprotein