Released glycans, analysis

Separation of the modified peptide from the released glycan may be accomplished using a Cig cartridge and the glycan then isolated from the unretained fraction by acid-catalyzed acetylation and organic/aqueous partition. Tandem mass-spectrometric analysis of the recovered peptide yields sequence information allowing location of the modified amino acid. 

In none of these reports was it attempted to recover the released glycans for further analysis, the purpose of the studies being to develop methods for identification of the glycosylation sites. 

For glycan analysis, the glycans are released by chemical or enzymatic methods. The prevailing analytical methods for glycan profiling involve some form of labeling... 

The lack of a pan-specific or broad specificity O-glycanase makes chemical methods the most appropriate approach for the non-specific release of intact O-linked oligosaccharides from glycoproteins for site analysis.219 The most common chemical methods employed for the release of O-linked glycans are based on (3-elimination. The methods were originally developed to release the glycans for... 

S. F. Wheeler and D. J. Harvey, Extension of the in-gel release method for structural analysis of neutral and sialylated A-linked glycans to the analysis of sulfated glycans Application to the glycans from bovine thyroid-stimulating hormone, Anal. Biochem., 296 (2001) 92-100. 

M. V. Novotny, Y. Huang, and Y. Mechref, Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis, Anal. Chem., 73 (2001) 6063-6069. 

A further consideration is the position of the sequon in relation to the primary structure of the protein. Statistical analysis of a large number of glycoproteins has indicated that the frequency of non-glycosylated sequons increases toward the C-terminus (Gavel and von Heijne, 1990). The critical distance appears to be 60 amino acid residues from the C-terminus when reduced glycan occupation occurs. This distance corresponds to the distance between the ribosome P-site and the active site of the OST and it has been hypothesized that the protein chain is not available for N-glycan attachment once it is released from the ribosome. However, this phenomenon of poor glycosylation efficiency toward the C-terminus does not appear to be universal for all proteins (Walmsley and Hooper, 2003). 

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