Evaluating the Performance of a New Synthetic Dipeptide Polymer for Glycopeptide Enrichment on a Hybrid Quadrupole Orbitrap Mass Spectrometer

Due to the significance of protein glycosylation in various biological processes, it is critical to characterize protein glycosylation accurately. In the past few decades, mass spectrometry (MS) has increasingly become a method to study protein glycosylation and made significant achievements in representing the roles of protein glycosylation in disease, such as cancer. In the general MS-based workflow, glycoproteins or glycopeptides usually require to be isolated from biological samples before MS analysis. Therefore, how to effectively enrich glycopeptides or glycoproteins from biological samples is crucial for successful characterization of protein glycosylation. Recently, a new material of dipeptide polymer (poly-PD@SiO2) was synthesized. In this study, the dipeptide polymer was used to enrich glycopeptides and a high resolution and high mass accuracy Hybrid Quadrupole Orbitrap mass spectrometer was utilized to evaluate the performance of enriching glycopeptides for this material. Firstly, fetuin and bovine serum albumin (BSA, as an interference protein) were mixed in different ratios to prepare a series of standard samples. Then, trypsin was used to digest samples. At last, glycopeptides were enriched by poly-PD@SiO2 before MS analysis. With the optimized enrichment procedures, poly-PD@SiO2 could successfully enrich glycopeptides from samples with resisting up to 100-fold BSA interference. However, for 10-fold BSA interference, samples enriched by hydrophilic interaction liquid chromatography (HILIC) were mainly non-glycopeptides. Therefore, it was demonstrated that poly-PD@SiO2 could provide more effective enrichment for glycopeptides in comparison with HILIC.