on 06-14-201402:02 AM - edited on 10-15-202111:07 AM by Closed Account
Viner R, Heemskerk AAM, Horn DM, Saba J, Bern M, Bush D, Santos M, Dewald H, Ivanov AR, Karger BL. ASMS 2014 Poster Purpose: To develop a middle-down workflow for the characterization of human
recombinant erythropoietin (rhEPO) glycoforms using a sheathless capillary
electrophoresis (CESI) and nanoflow liquid chromatography (nLC) based separation coupled to an Orbitrap mass spectrometer.
Methods: Reduced and alkylated rhEPO was digested with LysC. Digested rhEPO
was analyzed on Orbitrap-based instruments using CESI-MS or nLC/MS coupled with high resolution high mass accuracy (HR/AM) FT scanning of precursor and ETD or HCD MS2 fragment ions.
Results: To perform complete quantitative glycan site-specific mapping of rhEPO, we employed limited Lys-C digest to yield one glycosylation site per peptide fragment of 3–10 kDa size ideal for middle-down analysis. This enabled more complete sitespecific sequencing of glycoforms compared with top-down analysis. The rhEPO glycopeptides were well separated from non-glycosylated peptides by CESI. Glycoform resolution was mostly based on differences in the number of sialic acid residues. nLCseparation provided complementary information and better separation of large doubly glycosylated N- linked peptides.
Thermo Fisher Scientific, Leiden University Medical Center, ProteinMetrics, Barnett Institute Northeastern University.