Preparation of high-efficiency titanium ion immobilized magnetic graphite nitride nanocomposite for phosphopeptide enrichment

Background: Protein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains chal-lenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphoryla-tion prior to MS analysis.Results: In this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatog-raphy (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nano -composite had the advantages of a large specific surface (412.9 m2 g-1), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g- 1). Moreover, it presented satisfactory selectivity (alpha-casein:beta-casein: bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS.Significance: We successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.