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We have developed a versatile protocol for large-scale characterization of hydrophobic membrane proteins based on the periodic mesoporous organosilica (PMO) acting as both an extractor for hydrophobic substrate capture and a nanoreactor for efficient in situ digestion.With introduction of organic groups in the pore frameworks and the presence of hydrophilic silanol groups on the surface,PMO can be well dispersed into not only an organic solution to concentrate the dissolved membrane proteins but also an aqueous solution containing enzymes for sequential rapid proteolysis in the nanopores.The unique amphiphilic property of PMO ensures a facile switch in different phase systems to realize the multi-process of substrate dissolution,concentration,digestion,and MS measurement without cross influence effectively.Furthermore,this novel PMO-assisted protocol has been successfully applied for identification of complex membrane proteins extracted from mouse liver as proof of general applicability.