PMID: PMC3617342-2-5

 

    Legend: Gene, Sites

Title : Conventional Methods for Glycoproteomics

Abstract :

1. As reviewed elsewhere (36), conventional glycoproteomic methods involve the enrichment of glycoproteins (typically with lectins like ConA and wheat germ agglutinin ), cleavage of the glycans, and identification of the remaining peptide sequence
2. The most widely used method for detecting N-glycopeptides is digestion by PNGase F . PNGase F cleaves the GlcNAc molecule closest to the peptide (37)
3. After PNGase F treatment, formerly N-linked glycosylated peptides are identified based on the conversion of Asn to Asp (deamidation) in the consensus motif for N-linked glycosylation (sequence N-X(not P)-S/T)
4. This method has two major caveats
5. The first of these is a high false positive rate due to spontaneous deamidation
6. Asn-Gly sites , in particular, are prone to spontaneous deamidation (38–40)
7. To reduce false positives, PNGase F treatment is performed in 18O water, adding a larger tag of 2.99 Da
8. Importantly, all known glycosyltransferases that mediate N-linked glycosylation are supposed to recognize a consensus motif , and this consensus sequence for N-linked glycosylation must be taken into consideration (41)
9. 2) The second caveat is that after PNGase F cleavage, the released sugars can be analyzed separately, but the link to the identified peptides with deamidated amino acids is lost (42, 43)
10. Ideally, intact glycopeptides are analyzed directly via MS/MS even in complex biological samples

Output (sent_index, trigger, protein, sugar, site):

• 1. glycoproteins, , glycoproteins, -, -
• 10. glycopeptides, , -, -, glycopeptides
• 2. N-glycopeptides, , -, -, N-glycopeptides
• 3. glycosylated, , -, -, peptides

Output(Part-Of) (sent_index, protein, site):

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX