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Orbitrap_SciLib
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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‎10-15-2021 11:07 AM
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