N-linked and O-linked glycosylation are the two most common forms of glycosylation in mammals (45)
Only N-linked glycosylation was analyzed in the present study
Unlike N-linked glycosylation, O-linked glycosylation has no consensus site (46)
This makes the analysis of O-linked glycopeptides a more daunting task (47)
Lectins are widely used for glycoprotein enrichment
There are many types of lectins binding to different sugars, such as ConA (binds to α-d-mannosyl and α-d-glucosyl residues ) and wheat germ agglutinin (binds to GlcNAcβ1–4GlcNAcβ1–4GlcNAc- and N-acetylneuraminic acid)
Here we used only ConA as a proof of principle to demonstrate the complementary results of multiple glycoprotein identification methods
ConA is known to display nonspecific avidity for hydrophobic ligands such as certain domains of tropomyosin (48)
Furthermore, the standard protocol for the ConA glycoprotein enrichment kit is not optimized for cleanliness, and several known non-glycoproteins were also detected in the eluate samples
Sequential washes with low- and high-ionic-strength buffers before elution might have reduced this contamination (49)
Also, mixing different lectins would increase the coverage of the glycoproteome in biological samples (39)
Additional efforts are needed for a complete structural characterization of protein glycosylation; in particular, the quantitation of the occupancy rates and the identification of the glycan structure as complex/hybrid glycans cannot be discerned via our current MS approach