Recombinant V8 protease is the serine protease family and can specifically hydrolyzing glutamic acid or aspartic acid residues carboxy side peptide bonds. The Glu carboxy terminal peptide bond can be identified and cleaved in CH4COONH4 buffer (pH 7.8) and NH4HCO3 (pH 4.0 ).Identify and cleave the Glu or Asp carboxy terminal peptide bond in phosphate buffer (pH 7.8),and the hydrolysis rate of Glu was higher than that of Glu Asp. Recombinant V8 protease has activity between pH 4.0-10.0, and the optimum pH is 8.0-8.5. It can be used alone or in combination with other proteases when used in protein digestion, peptide profiling and peptide mass fingerprinting analysis. The inhibitors of V8 protease are diisopropylfluorophosphate (DFP), α2-macroglobulin and Nα-P-tosyl-L-lysine chloromethyl ketone (TLCK).