The invention provides a method for screening splitting sites and application of the method. The method comprises the following steps: S1, establishing a data writing a program by using a computer language, predicting that
intein is subjected to splicing reaction after being embedded in every two adjacent
amino acid residues in an
amino acid sequence and is
cut off, and forming a
protein database by using the
amino acid sequence formed by connecting adjacent
peptide fragments; s2, performing an experiment: inserting
intein sequence fragments into the
gene fragments, then performing
molecular cloning and translation to obtain
peptide fragments, detecting whether the
peptide fragments contain labeled amino acid sequences or not through
mass spectrometry, and comparing the peptide fragments with the
protein database to realize
verification of the
fracture sites. According to the method, a
protein database is constructed through
computer programming, then an experiment is carried out, and detection and
verification of splitting sites are realized through
mass spectrometry; final detection is achieved through
mass spectrum, high-
throughput screening is replaced, and the method is expanded to search for splitting sites of any
active protein.