Facile preparation of bifunctional adsorbents for efficiently enriching N-glycopeptides and phosphopeptides

In bottom-up strategy, specific enrichment of glycopeptides and phosphopeptides from complicated biological samples is a prerequisite for efficient identifying glycosylation and phosphorylation by mass spectrometry. Although there were a plethora of materials used as either hydrophilic interaction liquid chromatography (HILIC) or immobilized metal affinity chromatography (IMAC) adsorbents, even several bifunctional materials for simultaneous enrichment of glycopeptides and phosphopeptides, most of them are not easily commercialized as many other well-performing adsorbents due to the complicated preparation process. In our case, a one-step modification strategy was developed to prepare bifunctional adsorbents for HILIC and IMAC, employing O-phospho-l-serine as the modifier and poly(GMA-co-EDMA) microspheres, a kind of macroporous adsorption resin (MAR) with epoxy groups, as the matrix. The MARs were directly modified with O-phospho-l-serine under facile condition for HILIC strategy and further chelated with Ti⁴⁺ for IMAC strategy. A total of 522 unique N-glycopeptides and 442 unique N-glycosylation sites mapped to 275 N-glycoproteins was identified from HeLa cell proteins, showing excellent enrichment efficiency in HILIC. Additionally, 3141 unique phosphopeptides were unambiguously identified from 200 μg of digest of HeLa cell proteins, demonstrating great enrichment efficiency in IMAC. Moreover, these materials have been successfully applied in the analysis of multiple biological samples including human serum and milk, demonstrating their feasibility for real sample applications and potential business value.