Abstract: Protein glycosylation and phosphorylation stand as pivotal post-translational modifications (PTMs) in living organisms, profoundly influencing protein spatial conformation, biological activity, transportation, and localization. Moreover, they play a crucial role in specific biological processes such as cell growth and division, signal transduction, and metabolic pathways. In this study, we developed a dual-ligand hydrogen-bonded organic frameworks (HOFs) based on urea and phenylboronate salts for the simultaneous enrichment of glycopeptides and phosphopeptides from complex samples. The synthesized dual-ligand HOFs utilizes the N-H bond from the urea ligand and the oxygen atom from borate group of the 1,3,5-tris(4-phenylboronic acid)benzene ligand to form two sets of N-H···O hydrogen bonds, constructing a honeycomb-like two-dimensional network structure of HOFs. More importantly, the boron atom, with its vacant p-orbital, can form a B←N coordinate bond with the nitrogen in urea, thereby enhancing the structural stability of the HOFs. According to theoretical calculations, the functional sites of the HOFs exhibit a synergistic matching effect with both glycopeptides and phosphopeptides. We established a magnetic solid-phase microextraction-mass spectrometry (MSPE-MS) platform based on this dual-ligand magnetic HOFs. Using standard protein digests as a model, we investigated the dual enrichment capability of this method for glycopeptides and phosphopeptides, with limits of detection as low as 0.4 and 0.1 fmol/μL, respectively. Finally, this method exhibited effective separation performance for low-abundance phosphopeptides and glycopeptides in human saliva and serum samples, offering promising prospects for providing novel insights into high-throughput glycosylation and phosphorylation proteomics analysis.

Key words: hydrogen-bonded organic frameworks, dual-ligand, glycopeptides, phosphopeptides, magnetic solid-phase microextraction-mass spectrometry