Preparation of graphene–hafnium oxide composite for selective enrichment and analysis of phosphopeptides

Abstract

The reversible phosphorylation of proteins plays a crucial role in many regulatory processes. During the last decade, there has been considerable interest in the development of new methods for the identification of phosphorylation sites to allow for the comprehensive analysis of protein phosphorylation processes. However, the development of sensitive methods for the detection of trace quantities of phosphopeptides remains a significant challenge. In this study, we have prepared a novel graphene–hafnium oxide composite (GHOC) capable of enriching phosphopeptides and its application for the enrichment of phosphopeptides prior to their analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI – TOF/MS). The GHOC was prepared in a facile step using a hydrothermal reaction. The surface morphology of the resultant materials was analyzed in its compound-bound form by TEM, SAED and XRD. According to theses results, the GHOC had an increased surface area resulting from the template of graphene and the modification of hafnium oxide which possess high specificity toward phosphopeptides. Several complex samples (e.g. α- and β-casein, mixtures of β-casein and bovine serum albumin, and nonfat milk tryptic digest) were used to test the enrichment capability of the GHOC, and the results demonstrated that this material exhibited better selectivity towards mono- and multi-phosphorylated peptides compared with the graphene–TiO₂ composite (GTOC) and graphene–ZrO₂ composite (GZOC). Furthermore, MALDI-TOF/MS experiments revealed no interference from nonphosphopeptides. The results demonstrate that our newly developed GHOC shows high specificity for the enrichment of phosphopeptides from biological samples and could therefore be applied in the field of phosphoproteomics.