In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low‐abundance phosphopeptides

Abstract

Rationale Due to the dynamic nature of phosphorylation states and the low stoichiometry of phosphopeptides, it is still a challenge to efficiently capture phosphopeptides from complex biological samples before mass spectrometry analysis. Among the enrichment strategies, metal oxide affinity chromatography (MOAC) is one of the most widely used and the one with the most potential. It is based on reversible Lewis acid–base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides. Methods A novel MOAC affinity probe, denoted as G@PDA@ZrO2, was successfully synthesized by in situ grafting ZrO2 onto the surface of graphene (G) modified with polydopamine (PDA). The novel MOAC probe thus obtained was used for phosphopeptide enrichment. Results This novel MOAC affinity probe when used to selectively enrich phosphopeptides from standard protein digest solutions exhibited a high selectivity (β‐casein:bovine serum albumin = 1:1000), a low detection limit (4 fmol), and a high loading capacity (400 mg/g). At the same time, the experimental results proved that G@PDA@ZrO2 had great recyclability (five cycles), stability, and reproducibility. Subsequently, G@PDA@ZrO2 was applied to enrich phosphopeptides from human saliva and human serum, in which 25 and 4 phosphopeptide peaks, respectively, were detected. Conclusions This novel MOAC affinity probe (G@PDA@ZrO2) showed good performance in enriching phosphopeptides. Thus, G@PDA@ZrO2 has good potential in phosphopeptidomics analysis.

Recent trends in sorbents for bioaffinity chromatography

“…Studies on MOAC, started with the use of titanium dioxide (TiO 2 ) for the enrichment of phosphopeptides, and continued with the development of various metal oxides such as zirconium dioxide (ZrO 2 ), magnetite (Fe 3 O 4 ), tin oxide (SnO 2 ), and cerium oxide (CeO 2 ) [23,70,[72][73][74][75][76][77][78][79]. The selectivity of metal oxides against phosphopeptides varies according to their properties such as surface area, pore structure, and isoelectric point.…”

“…Bidentate chelate formation in the interaction of phosphoproteins with CeO 2 microspheres as a new MOAC sorbent [79] Furthermore, metal oxides based materials such as ZrO 2 and CeO 2 were used to enrich phosphopeptides from complex biological samples in the past decade [73,77,79]. A magnetic porous MOAC sorbent (PI-FexOy-ZrO 2 ) was designed by Wang et al, through one-step strategy to selectively enrich phosphopeptides [77].…”

“…PI-FexOy-ZrO 2 nanocomposite performed better in phosphopeptide adsorption than commercial titanium dioxide nanoparticles [77]. Wang et al grafted ZrO 2 onto the surface of graphene to prepare a MOAC affinity probe (G@PDA@ZrO 2 ), which was used for selective enrichment of phosphopeptides from standard protein digest solutions (β-casein: BSA = 1:1000), also displayed a low detection limit (4 fmol), and high loading capacity (400 mg/g) [73].…”

“…The use of carbon‐based materials and graphene supporting the metal oxide (MO) nanoparticles are alternative ways to prevent the agglomeration of nanoparticles and therefore their loss during separation. [23, 45,73–75, 77,78,87,88].…”

Facile Preparation of Chitosan‐modified Mesoporous Titanium Dioxide Film on Fused‐silica Capillary for Selective Enrichment of Phosphopeptides

“…Protein phosphorylation is a phosphoryl transfer reaction catalyzed by protein kinases. Phosphorylation of proteins takes an important part in regulating many complicated biological processes including cellular recognition, signal transduction, individual metabolism, cell proliferation and differentiation [1–5] . It is necessary to identify and analysis phosphorylation sites for protein.…”