Improved Characterization and Quantification of Complex Cell Surface N-Glycans

MSextractcroppedN-Glycosylation is a common protein post-translational modification occurring on asparagine residues of the consensus sequence asparagine-X-serine/threonine, where X may be any amino acid except proline. Protein N-glycosylation takes place in the endoplasmic reticulum (ER) as well as in the Golgi apparatus.

Approximately half of all proteins typically expressed in a cell undergo this modification, which entails the covalent addition of sugar moieties to specific amino acids. There are many potential functions of glycosylation. For instance, physical properties include: folding, trafficking, packing, stabilization and protease protection. N-glycans present at the cell surface are directly involved in cell−cell or cell−protein interactions that trigger various biological responses.

The standard method used to profile the N-glycosylation pattern of cells is glycoprotein isolation followed by denaturation and/or tryptic digestion of the glycoproteins and an enzymatic release of the N-glycans using PNGase F followed by analysis mass spec. This method has been reported to yield high levels of high-mannose N-glycans that stem from both membrane proteins as well as proteins from the ER.(1,2)

For those researchers interested in characterizing only cell surface glycans (i.e.,  complex N-glycans)  a recent reference has developed a model system using HEK-292 cells that demonstrates a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells (3). The method involves standard centrifugation followed by enzymatic release of cell surface N-glycans. When compared to the standard methods the detection and quantification of complex-type N-glycans by increased their relative amount from 14 to 85%.

  1. North, S. J. et al. (2012) Glycomic analysis of human mast cells, eosinophils and basophils. Glycobiology. 2012, 22, 12–22.
  2. Reinke, S. O. et al. (2011) Analysis of cell surface N-glycosylation of the human embryonic
    kidney 293T cell line.
    J. Carbohydr. Chem.  30, 218–232.
  3. Hamouda, H. et al. (2014) Rapid Analysis of Cell Surface N‑Glycosylation from Living Cells Using Mass Spectrometry. J of Proteome Res. 13, 6144–51.

Therapeutic Manipulation of N-glycan Branching: Promise in the Fight against MS?

Multiple Sclerosis is characterized by inflammatory demyelination and degeneration of the nervous system.

Multiple Sclerosis (MS) is a horrible, debilitating disease that affects an estimated 3 million people world wide. My friend “Liz” is one of those 3 million. When I first met Liz, she was a bright bubbly young woman who loved crafts and entertaining. She had a huge room in her basement filled with rubber stamps, paper and other craft supplies. The first Christmas I knew her, she and her husband had four Christmas trees in their house. Liz decorated each tree with a different theme. Then abruptly she stopped appearing at the social functions she never missed. A year or so later came the news that she had been diagnosed with Multiple Sclerosis. Continue reading