For O18 labeling based comparative proteome analysis,enzyme-catalyzed protein digestion and peptide labeling are two crucial sample preparation steps for protein identification and quantification.However,traditional in-solution protein digestion and enzymecatalyzed O18 labeling not only might suffer from time-consuming,sample loss and incomplete labeling due to back exchange,but also it was difficult to integrate traditional in-solution digestion protocol with HPLC-MS for on line protein analysis,which might result in irreproducibility and low analysis throughput.