The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS).  Firstly, the molecular mass of deglycosylated protein was measured.  Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications.  Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with ~80% occupancy; N103, N448 and N184 (~15% occupancy) were glycosylated with complex-type oligosaccharides.  LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.