Abstract: OBJECTIVE To analyze the peptide mass mapping and localization of disulfide bonds of recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF) by using mass spectrometry combined with ultra-performance liquid chromatography. METHODS The bulk of rhGM-CSF was denatured, reduced, alkylated and finally digested by the enzyme of trypsin and V₈ respectively to analyse the peptide mass mapping. The bulk was denatured, alkylated and digested by V₈ and V₈ with trypsin respectively to identify the sites of disulfide bonds. RESULTS Fourteen matched peptides were found in the peptide mass mapping of rhGM-CSF digested by trypsin and eighteen matched peptides were found digested by V₈. The fraction of coverage was both 100.0%. The site of disulfide bond Cys55-Cys97 was identified by digestion of V₈, and the types of disulfide bonds Cys55-Cys97 and Cys89-Cys122 were identified by digestion of V₈ with trypsin. CONCLUSION The peptide mass mapping analysis and localization of disulfide bonds using LC-MS/MS can be used for the quality control of recombination protein.