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In this report, laser-assisted proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly with the assistance of laser irradiation (808 nm) both in sealed transparent Eppendorf tubes and on the spots of a stainless steel MALDI plate. Besides, laser irradiation (808 nm) was employed to enhance the efficiency of in-gel tryptic digestion of proteins separated by SDS-PAGE. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of standard proteins and real sample. It was demonstrated that laser irradiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to seconds. The digests were characterized by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF-MS) with sequence coverage of 42% (BSA), 89% (myoglobin) and 76% (cytochrome c), respectively. The suitability of the new digestion approach to complex proteins was demonstrated by digesting rat brain. The present proteolysis protocol is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.