Detailed knowledge on glycan composition and their site-specific distribution within a specific glycoprotein is crucial for understanding their complex biological functions. Reliable glycopeptide identification providing concomitant details on both, the glycan and peptide moieties still remains challenging, also because glycopeptides are highly heterogeneous molecules. Defined, synthetic glycopeptides offer a unique opportunity to investigate and validate glycoproteomics sample preparation and analytical workflows and develop novel methods.
We have developed a novel, simplified approach to purify and produce a panel of glycosylated Fmoc-protected Asparagine amino acids carrying N-linked glycans with various structures. These building blocks were subsequently used in standard solid phase glycopeptide synthesis to generate a synthetic glycopeptide library containing >100 glycopeptides and their unglycosylated counterparts.
First, a novel, simple, fast and cost-effective technique for HILIC (hydrophilic interaction chromatography) based glycopeptide enrichment ("Drop-HILIC") was developed. Drop-HILIC was used to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type HILIC) glycopeptide enrichment. We found that glycopeptide enrichment efficiency primarily relied on the applied mobile phase, but we also found that even minimal glycopeptide structure/composition differences already affected ZIC-HILIC enrichment .
Glycopeptide ionisation efficiency was investigated using CaptiveSpray Nanobooster™. This allows overcoming possible glycopeptide enrichment biases  and reduced glycopeptide ionisation efficiacy . The use of the CaptiveSpray Nanobooster™ itself already resulted in ~5-fold increase in glycopeptide signal intensities compared to conventional CaptiveSpray nano ESI ionisation without any changes to the sample preparation workflow or MS hardware.
Finally, a panel of synthetic glycopeptides was used to evaluate how glycan size and glycosylation site location within a peptide influence ETD fragmentation efficiency and successful MASCOT assignment. The number and quality of assignable peptide backbone fragments was significantly depending on glycan size, the position of the modification within a peptide sequence and the individual precursor m/z.