N-glycosylation is one of the most prominent posttranslational protein modifications involved in many cellular functions including cell-cell and receptor-ligand interactions, immune response, apoptosis, and pathogenesis of many diseases. Asparagines, typically in the consensus sequence motif N-!P-[S|T] (where !P signifies any amino acid except proline), can be glycosylated with widely varying glycan structures. This co-translational modification takes place predominantly in the lumen of the ER and Golgi apparatus. The modification is thought to occur on secreted molecules, the extracellular part of plasma membrane proteins, and the luminal part of proteins in compartments of sub-cellular organelles such as the endoplasmic reticulum and the Golgi apparatus, endosomes, and lysosomes.

 

To detect low abundant N-glycosylated proteins or peptides in complex mixtures among the large excess of their non glycosylated counterparts with LC-MS techniques, specific enrichment methods have to be applied, most commonly based on lectin affinity or chemical linkage of the sugar moiety to surfaces. In our laboratory, we extensively use methods based on lectin affinity of glycopeptides whereby the enrichment step is performed using “filter aided sample preparation” (FASP). After enzymatic digestion of the protein sample, the resultant peptides are mixed with lectins on spin-filters. The glycopeptide that are retained can later be released by a universal deglycosylating enzyme (i.e., PNGase F). The former site of glycosylation can be recognized as during deglycosylation the asparagine residue is deamidated resulting in a mass increase of 0.9840 Da. To distinguish deglycosylation from spontaneous deamidation and to add confidence in the site assignment, deglycosylation can be performed in 18O-water, leading to a mass shift of 2.9883 Da.

 

• Wisniewski et al, Nat Methods (2009)
• Zielinska et al, Cell (2010)
• Ostasiewicz P. et al, J. Proteome Res. (2010)
• Wisniewski J. et al, Anal Biochem. (2011)

 

 

Recommended protocol:

This is available at http://www.biochem.mpg.de/mann/fasp/n_glyco_fasp/index.html

 

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