Analysis of intact erythropoietin and novel erythropoiesis-stimulating protein by capillary electrophoresis-electrospray-ion trap mass spectrometry

Electrophoresis
Estela GiménezVictoria Sanz-Nebot

Abstract

Recombinant human erythropoietin (rHuEPO) and novel erythropoiesis-stimulating protein (NESP) were analyzed by CE-ESI-MS using an IT as analyzer. The IT parameters were optimized by direct infusion of solutions of different intact proteins (myoglobin, transferrin, alpha1-acid glycoprotein and fetuin) with different degrees of glycosylation (from 0 to 35% w/w). Two physically adsorbed capillary coatings from UltraTol Pre-Coats (low normal (LN) and high reverse (HR)) were evaluated for the separation of rHuEPO and NESP glycoforms by CE-ESI-IT-MS. The results obtained with the neutral LN coating suggest that an IT mass spectrometer enables identification of the main glycoforms of a complex glycoprotein such as rHuEPO. Although LN provided acceptable glycoform resolution for rHuEPO, the separation obtained for NESP was less significant due to the higher microheterogeneity of this glycoprotein. Reproducibility studies confirmed the lack of stability and bleeding of the LN coating, which caused problems with MS detection, such as a dramatic loss of sensitivity and the presence of peaks in the mass spectra corresponding to molecular ions in the coating. In contrast, the cationic HR coating gave faster but poorer glycoform separations ...Continue Reading

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Citations

Oct 20, 2009·Analytical and Bioanalytical Chemistry·C HuhnG W Somsen
Jul 24, 2013·Analytical Biochemistry·James M HempeStuart A Chalew
Apr 26, 2014·Forensic Science International : Synergy·Isabelle KohlerJulie Schappler
Jul 22, 2009·Electrophoresis·Youssef AlahmadMyriam Taverna
Jul 15, 2011·Rapid Communications in Mass Spectrometry : RCM·Estela GiménezVictoria Sanz-Nebot
Jun 1, 2012·Electrophoresis·Angelina TaichribChristian Neusüss
Aug 21, 2016·Analytica Chimica Acta·Giorgio Gianini MorbioliEmanuel Carrilho
Apr 23, 2010·Rapid Communications in Mass Spectrometry : RCM·Marta Borges-AlvarezVictoria Sanz-Nebot
Nov 3, 2020·Journal of Separation Science·Shannon L ThomasKatarína Maráková
Nov 2, 2019·Journal of Pharmaceutical and Biomedical Analysis·Julien CamperiNathalie Delaunay

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