Detailed information on glycoprotein primary structure is a prerequisite for understanding their diverse functions. Aspects such as glycosylation micro- and macroheterogeneity (1) are nowadays mostly determined using MS and tandem MS approaches (2). Conquering the final frontiers in the glycospace requires also a solid understanding on glycoprotein and glycopeptide related sample preparation and MS analysis aspects. Synthetic glycopeptides offer a unique opportunity to investigate and validate glycoproteomics analyses and develop new methods.
We have developed novel and simplified approaches to produce N-glycan carrying amino acids for solid phase peptide synthesis. In combination with targeted chemo-enzymatic glyco-modifications these building blocks allowed us to establish a well-defined glycopeptide library containing >100 glycopeptides plus their unglycosylated counterparts. This library has been used to systematically assess glycoproteomics aspects such as HILIC glycopeptide enrichment, collision stepping CID, effective ionisation and ion-mobility glycoproteomics.
This capacity allowed us to quantify and determine the ionization suppression effect glycopeptides are experiencing compared to their unglycosylated counterparts (3). CaptiveSpray Nanobooster™ ionisation, however, can effectively enhance their ionization, providing access to compounds hardly detected by other ESI ionisation techniques. This glycopeptide library also enabled us to optimise CID/HCD/ETD, fragmentation by improving data quality for software assisted glycopeptide assignment. These optimised conditions were also validated on biologically relevant samples such as the entire panel of human Immunoglobulins (4). The solvent effect on ZIC-HILIC glycopeptide enrichment was systematically evaluated, revealing a tremendous solvent-dependability (5). These synthetic N-glycopeptides were also the key to develop innovative ion-mobility based techniques providing glycan structure information on sialic acid linkage directly from glycopeptide tandem MS data (6), making a huge leap towards site specific glycan structure analyses going beyond compositional data. This toolkit provides a solid basis to find answers to the functional role of protein glycosylation in health and disease.