Abstract: To investigate the influence of missed cleavage numbers of trypsin for database searching results, Cytochrome C and abscisic stress ripening-like protein were identified by MALDI-TOF/TOF mass spectrometry. Two and four tryptic peptides with double missed cleavage were found in the digests of cytochrome C and of abscisic stress ripening-like protein, respectively. It was found that two missed cleavage will happe when two lysine residues neighbor to each other or lysines and arginines neighbor with an aspartic acid or glutamic acid residue. It was observed that this phenomenon was related to histine-lysine-isoleunine/alanine sequence. In this study, the certainty of protein identification rather than protein coverage was significantly improved when tryptic peptides with double missed cleavage sites were identified, which indicate that false negative of peptide identification will happen when the number of missed cleavage site was set to 1.