Primary structure determination of recombinant human erythropoietin by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry

A series of method for the primary structure determination of recombinant human erythropoietin (rhEPO), a recombinant glycoprotein, by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) was developed which includes the methods of the measurement of molecular weight, content of sialic acid and carbohydrate in rhEPO etc. Based on the developed methods, four kinds of rhEPO coming from different manufactories were determined.. The coverage of PMF of all rhEPOs reached 98% by optimization of digestion condition, and N-glycan site and two disulphide bridges in the rhEPOs were determined successfully. Molecular weight of four samples are (27754. 1 +/- 27. 4.) Da, (27941.4 +/- 23. 2)Da, (27682.5 +/- 22. 0) Da and (27914. 7 +/- 22. 5) Da; content of sialic acid are 5. 0%, 4.8%, 5.6% and 5.4%; and content of carbohydrate are 34.3%, 34.7%, 34.1% and 34.6%. Moreover, the difference of the glycan structure type on among these four rhEPOs was probed and discussed.