Glycoproteomic analysis – opportunities and threats

Against the background of tremendously complex proteomes the presence of a heterogeneous modification on possibly every second protein is supposed to complicate analysis even more. Glycosylations are indeed involved in many essential functions like cell-cell interactions, protein processing and furthermore pathological conditions like cancer - yet their analysis within comprehensive cellular systems is supposed to be a major future challenge.

In order to supply a comprehensive glycosylation analysis different types of information have to be assembled: The site of modification, the exact glycans structures on the protein and their attachment sites. We have been working on the elucidation of glycosylation sites by establishing dedicated mass spectrometric workflows for the assignment of N-glycosylation sites e.g. in human platelets. Applied to whole cell digests, these workflows enable the enrichment of glycosylated peptide subsets e.g. by charged-based enrichment and parallel analysis of hundreds of glycosylation sites. Beside the benefits of assigned modification sites for the research community, these methods also allow for identification of low abundant membrane receptors due to removal of high abundant but non-glycosylated peptides. Furthermore, they hopefully offer the potential for relative quantitation of glycosylation sites in the future.

However, the assignment of glycosylation sites by mass spectrometry is not unbiased. It is hindered by analytical and biological obstacles like e.g. natural or artificial deamidation. Therefore, it is mandatory to provide correct estimates of false positive identification rates in order to assess the quality of large scale datasets.