Abstract: A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti4+affinity chromatography (Ti4+-IMAC) was proposed, and applied in the global profiling of phosphorylated membrane proteome of neuroblastoma SH-SY5Y cells. The fraction of membrane proteins was extracted by ultra speed centrifuge, followed by washing with 1 mol/L sodium chloride and 0.1 mol/L sodium carbonate. For digestion, chymotrypsin and pepsin with broader specificity were used as complementary enzymes to trypsin. The phosphopeptides were then selectively enriched by monolithic Ti4+-IMAC column, and analyzed by nanoflow high performance liquid chromatography and mass spectrometry. A total of 43 phosphoproteins were identified, among which 14 proteins were located on the membrane. All these results demonstrated that the proposed strategy might be promising to promote the in-depth study of neuroblastoma and discover the candidate biomarkers.

Key words: immobilized Ti4+affinity chromatography, membrane protein, multi-enzyme digestion, organic-inorganic hybrid silica monolith, human neuroblastoma, phosphopeptide enrichment