In order to establish a method for analyzing the distribution and localization of disulfide in recombinant protein by specific protease digestion combined with MALDITOF/MS. The pET22b (+) prokaryotic system was harnessed to generate recombinant protein of human plasminogen kingle (rhK5) which was purified by Ni

2+

-NTA resin affinity chromatography. SDS-PAGE and Western blot analysis were used to identify the recombinant protein. The disulfide bridging conformation and dimensional structure of rhK5 were predicted by 3DJIGSAW Comparative Modeling Server (UK) and the disulfide bond distribution of rhK5 was confirmed by orthogonal enzymatic digestion and MALDITOF/MS. The secondary structure of rhK5 was analyzed by circular dichroism. Enzymatic digestion and MALDI-TOF/MS analysis demonstrated the presence of disulfide bridges among Cys462Cys541

Cys483Cys524 and Cys512Cys536 in rhK5. Circular dichroism showed that rhK5 adopted the shape characteristic of a β-sheet. Our studies provide methodological basis for analyzing the pairing and distribution of disulfide bond of genetic engineering recombinant proteins.