Design and synthesis of an immobilized metal affinity chromatography and metal oxide affinity chromatography hybrid material for improved phosphopeptide enrichment

Abstract

Reversible phosphorylation of proteins is one of the most important post-translational modifications, while the detection of phosphopeptides is difficult due to their low abundance and the signal suppression of nonphosphorylated peptides. Therefore, selective enrichment of phosphopeptides from highly complicated mixtures is vital for MS-based phosphoproteome analysis. Despite various strategies have been developed, there is no single method that is capable of providing full coverage of the whole phosphoproteome. Metal oxide affinity chromatography (MOAC) enrichment preferably singly phosphopeptides, whereas immobilized metal affinity chromatography (IMAC) enrichment bias towards multiply phosphopeptides. In this study, first example of IMAC and MOAC hybrid material, Fe₃O₄@nSiO₂@mSiO₂/TiO₂-Ti⁴⁺ nanoparticles were successfully synthesized for the enrichment of phosphopeptides with the aim to combining their advantages for enrich both mono- and multi-phosphorylated species. The TiO₂ was firstly coated on the surface of mesoporous silica and then grafted with 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to chelate Ti⁴⁺ ions. This novel type of hybird material with high surface areas (179.3m²/g) exhibited good adsorption capacity (133mg/g) towards standard tryptic digest of β-casein and the method based on this material also showed good sensitivity (4pmol). The synthesized Fe₃O₄@nSiO₂@mSiO₂/TiO₂-Ti⁴⁺ microspheres were further used to selectively enrich phosphopeptides from complex biosamples, seven mono-phosphopeptides and eight multi-phosphopeptides were successfully enriched from nonfat milk which is much better than single IMAC or MOAC strategy. Those results indicated that Fe₃O₄@nSiO₂@mSiO₂/TiO₂-Ti⁴⁺ microspheres have potential applications in MS-based phosphoproteomics to enlarge phosphoproteomics coverage and this work was expected to open up a promising strategy which combined the advantages of various methods in one material for effective enrich phosphorylated peptides.

Keywords: Immobilized metal affinity chromatography; Mass spectrometric analysis; Phosphopeptides; Phosphoproteomics coverage; Titanium oxide.