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Reputable Mentor II
Reputable Mentor II
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.
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Last update:
‎10-15-2021 11:07 AM
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