Porous coordinated polymers/chitosan composite monolith with directional pore structure for highly efficient mono−/multi−/global phosphopeptide enrichment

Preferential enrichment of phosphopeptides with different numbers of phosphorylation sites is crucial to investigate the modulation of signal transduction pathways of a graded protein kinase or phosphatase signal. Here, zirconium phosphonates chitosan monoliths (ZrPCMs) with varying compositions were constructed through a salt-forming reaction and bidirectional freezing strategy. These composite monoliths exhibited differential coordination abilities for mono- and multi-phosphopeptides without the need for centrifugation or a magnetic field during enrichment process. By the utilization of different ZrPCMs and control of loading amount of sample, selective enrichment of mono- or multi-phosphopeptides was realized. At high loading amounts, excess phosphate groups on ZrPCM-1 inhibited mono-phosphopeptides with chelating with Zr (IV), resulting in the preferential enrichment of multi-phosphopeptides. In contrast, ZrPCM-2 enabled the enrichment of global phosphopeptides due to its balanced phosphate group and Zr(IV) loading, avoiding selective preferences under extreme conditions. At low loading amounts, the availability of sufficient Zr (IV) on ZrPCM-3 facilitated the preferential enrichment of mono-phosphopeptides due to their lower steric hindrance and more stable binding, while the adsorption ability for multi-phosphopeptides was reduced owing to local charge accumulation effects and electrostatic repulsion. These results were verified not only in standard protein but also in complex biological samples involving nonfat milk and mouse liver. Consequently, this present work developed promising ZrPCMs materials and isolation strategy of mono-, multi-, or global phosphopeptides for phosphoproteome research.