Assigning N-glycosylation sites of glycoproteins using LC/MSMS in conjunction with endo-m/exoglycosidase mixture

Zaneer M. Segu, Ahmed Hussein, Milos V. Novotny, Yehia Mechref

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Abstract

The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, unequivocal assignment of protein glycosylation remains to be a daunting task, prompting continuous demands for the development of sensitive and cutting-edge analytical approaches. aβ-N- Acetylglucosaminidase (endo-aβ-GlcNAc-ases, Endo-M) is an endoglycosidase capable of hydrolyzing N,N-diacetylchitobiose moiety in N-linked oligosaccharides bound to the asparagine amino acid residue in various glycoproteins. An attractive feature of this enzyme is its ability to cleave the N,N-diacetylchitobiose moiety while leaving an N-acetylglucosamine residue bound to the protein. This enzyme is also known to be inactive in the presence of core fucose residue linked to the reducing-end N-acetylglucosamine residue (GlcNAc). Here, we describe an approach capitalizing on these features of Endo-M to (a) determine the glycosylation sites of proteins and the occupancy of these sites, and (b) determine the attachment sites of fucose residue containing N-glycans. The latter is important because of its biological implications. Tryptically digested glycoproteins, which were subjected to Endo-M treatment, were analyzed by LC-MS/MS. Systematic evaluation of the activity of Endo-M toward different glycan structures indicated a dependence of enzyme activity on the complexity of the glycan structures. Efficient release of N-glycans using Endo-M is only achieved through the inclusion of a battery of exoglycosidases to reduce the complexity of the attached glycans and subsequently prompt an effective enzymatic release. Upon Endo-M/exoglycosidase treatment of tryptically digested glycoproteins, glycosylated sites retain GlcNAc residue. The resulting peptides with GlcNAc residues attached to the glycosylation sites are easily assigned through LC-MS/MS analysis and subsequent database searching of the generated tandem MS of such entities. Comparing the LC-MS/MS results of the tryptic digest of glycoproteins treated with PNGase F and Endo-M/exoglycosidases allowed the assignment of core fucose residues to N-glycan reducing-ends. The detection of glycosylation sites only in the tryptic digest of PNGase F treated samples suggested core fucosylation of the attached N-glycans to such sites. This strategy was initially validated using model glycoproteins. It also proved to be useful in determining the glycosylation sites of blood serum glycoproteins.

Original languageEnglish
Pages (from-to)3598-3607
Number of pages10
JournalJournal of Proteome Research
Volume9
Issue number7
DOIs
StatePublished - Jul 2 2010

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Keywords

  • Endo M
  • Exoglycosidases
  • Glycoprotein
  • Glycosylation sites
  • PNGase F

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