Study Of Glycosylation Sites In Recombinant Human Erythropoietin(Rhepo) Using Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

Determination doping especially peptide hormones is very difficult. Recombinant human erythropoietin erythropoietin (rhEPO) is a typical glycoprotein, containing three N-linked glycosylation sites and one O-linked glycosylation site. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry is good at qualitative structural analysis due to its high resolution, high sensitivity, wide mass range, and multiple dissociation techniques that precede the conventional dissociation techniques in mass spectrometric analysis. FT-ICR MS allows the determination of the position of the post-translational modifications, as well as, the structure of the modification, and enables the detection of different fragmentations of peptides or proteins at lower levels.rhEPO is digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Glycopeptide powder is purified by liquid chromatography, and then freeze-dried. The FT-ICR MS analytical strategy combines advanced CAD with ECD technology to identify O-linked glycosylation sites and glycosylation structures of rhEPO. Furthermore, study was carr ied out on the tw o different valence states of O-linked glycopeptides by ECD modes. This method provides a simple, rapid and effective way for the detection and identification of the O-linked glycosylation sites in rhEPO. This method not only has good prospects in the determination of peptide stimulates abuse, but also has high practical value in the accurate measurement of low protein content in clinical samples.