Search Methods for Identification of Sites of N-glycan Attachment
N-glycans are attached to the side chain of the amino acid Asparagine, but only when that Asparagine residue (N) is present in the tripeptide consensus sequence N-X-S/T where X is any amino acid except Proline and S and T are Serine and Threonine respectively. The presence of this consensus sequence is no guarantee of N-glycosylation but if it happens then this is where it will be located. So how can we find this?
Well, if there is only one potential N-glycosylation site in the molecule then there is nowhere else it can be! However, for those molecules that have more than one consensus site we have some options at the protein analysis level we can use.
Firstly, peptide mapping can be used to identify glycosylation sites, both occupied and unoccupied. With the careful selection of proteolytic enzyme or digestion strategy, the glycopeptides (or predicted glycopeptides) can be identified individually in an LC-MS experiment. The same chromatography can then also be used as a fraction collection process to collect the separated glycopeptides for subsequent identification of what N-glycosylation populations are where, because it is highly likely that the distribution of N-glycans will not be the same at each glycosylation site.
Degree of occupancy at specific sites of glycosylation
So, the above helps us identify the sites of glycosylation, but we can actually go a step further and assess the degree of occupancy at the individual glycosylation sites. This is achieved using the enzyme PNGase F, which releases N-glycans from the protein backbone and in so doing converts the glycosylated Asparagine residue to Aspartic acid. This amino acid is 1Da heavier than Asparagine and so the modified peptide can readily be identified in a mass spectrometer during an on-line LC/MS peptide mapping analysis where PNGase F has been used to release N-glycans.
A comparison of the peak areas of the peptide with Asparagine vs Aspartic acid then allows, in a relative abundance sense, a determination of the degree of occupancy of the glycosylation site. This doesn’t take into account any differences in ionization efficiency between the two peptides however, but for comparative purposes this effect is the same for all samples and thus cancels out.