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 wash-lng 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.