An efficient strategy with a synergistic effect of hydrophilic and electrostatic interactions for simultaneous enrichment of N - and O -glycopeptides.

N - and O -glycosylation modifications of proteins are closely linked to the onset and development of many diseases and have gained widespread attention as potential targets for therapy and diagnosis. However, the low abundance and low ionization efficiency of glycopeptides as well as the high heterogeneity make glycosylation analysis challenging. Here, an enrichment strategy, using Knoevenagel copolymers modified with polydopamine-adenosine (denoted as PDA-ADE@KCP), was firstly proposed for simultaneous enrichment of N - and O -glycopeptides through the synergistic effects of hydrophilic and electrostatic interactions. The adjustable charged surface and hydrophilic properties endow the material with the capability to achieve effective enrichment of intact N - and O -glycopeptides. The experimental results exhibited excellent selectivity (1 : 5000) and sensitivity (0.1 fmol μL ^-1 ) of the prepared material for N -glycopeptides from standard protein digest samples. Moreover, it was further applied to simultaneous capturing of N - and O -glycopeptides from mouse liver protein digests. Compared to the commercially available zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) material, the number of glycoproteins corresponding to all N - and O -glycopeptides enriched with PDA-ADE@KCP was much more than that with ZIC-HILIC. Furthermore, PDA-ADE@KCP captured more O -glycopeptides than ZIC-HILIC, revealing its superior performance in O -glycopeptide enrichment. All these results indicated that the strategy holds immense potential in characterizing N - and O -intact glycopeptides in the field of proteomics.