Abstract: OBJECTIVE To compare the performance of four kinds of dissociation methods in characterizing the peptide sequence of oxytocin by Orbitrap high-resolution mass spectrometry. METHODS The separation was performed on a fused silica capillary column(packed with C₁₈ resin) with the mobile phase consisting of 0.1% formic acid aqueous solution(mobile phase A) and 0.1% formic acid aqueous solution-acetonitrile(20:80)(mobile phase B) by gradient elution. The mass spectrometry was operated in the parallel reaction monitoring(PRM) under positive ion mode using electrospray ion source. Four kinds of dissocation methods, namely, collison induced dissociation(CID), higher-energy collision dissociation(HCD), electron transfer dissociation(ETD) and electron transfer/higher-energy collision dissociation(EThcD) were used to characterize the peptide sequence of oxytocin. RESULTS EThcD achieved the complete sequence coverage by producing the most abundant sequence informative ions related to peptide backbone. ETD achieved the moderate peptide sequencing, and ETD was slightly better than HCD, while CID performed the worst. Also, EThcD, ETD and HCD all produced characteristic ions relecting the disulfide bond in oxytocin, however, these ions were absent in CID mass spectra. CONCLUSION EThcD outperforms all other three dissociation methods in oxytocin sequence coverage. HCD and ETD can be complementary dissociation methods for peptide characterization. Using CID alone seems to be not adequade for oxytocin peptide sequencing. EThcD is a promising technique in peptide sequence for cyclopeptide with disulfide bond.