MS Platform for Erythropoietin Glycome Characterization

Seo, Youngsuk; Kim, Unyong; Oh, Myung Jin; Yun, Na Young; An, Hyun Joo

doi:10.5478/msl.2015.6.3.53

Cited by 8 publications

(3 citation statements)

References 44 publications

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“…It has been shown that the glycan composition of the biopharmaceuticals is critically depending on the presence and activity of different glycosyltransferases, concentration of substrates, cell lines, and cell culture conditions, making the production of therapeutic glycoproteins challenging . EPO is highly heterogeneous with a variety of glycosylation patterns as well as post-glycosylational modifications, such as sialylation by N -acetylneuraminic acid (Neu5Ac) or N -glycolylneuraminic acid (Neu5Gc), phosphorylation, and O-acetylation . These patterns have a significant impact on the drug efficiency and adverse drug effects in EPO-treated patients.…”

Section: Introductionmentioning

confidence: 99%

“… 5 EPO is highly heterogeneous with a variety of glycosylation patterns as well as post-glycosylational modifications, such as sialylation by N -acetylneuraminic acid (Neu5Ac) or N -glycolylneuraminic acid (Neu5Gc), phosphorylation, and O-acetylation. 6 These patterns have a significant impact on the drug efficiency and adverse drug effects in EPO-treated patients. As an example, increases of the Neu5Gc containing glycoproteins can lead to immunogenic or inflammatory responses in patients.…”

Section: Introductionmentioning

confidence: 99%

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An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

et al. 2021

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The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for Oglycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

et al. 2021

View full text Add to dashboard Cite

show abstract

“…5 EPO is highly heterogeneous with a variety of potential glycosylation patterns as well as post-glycosylational modifications, such as sialylation by N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), phosphorylation and O-acetylation. 6 These patterns have a significant impact on the drug efficiency and adverse drug effects in EPO-treated patients. As an example, increases of the Neu5Gc containing glycoproteins can lead to immunogenic or inflammatory responses in patients.…”

Section: Introductionmentioning

confidence: 99%

An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry

Guan

Zhang

Gaikwad

et al. 2021

Preprint

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The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

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NEGATIVE ION MASS SPECTROMETRY FOR THE ANALYSIS OF N‐LINKED GLYCANS

Harvey

2020

Mass Spectrometry Reviews

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N‐glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure‐revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N‐linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix‐assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique. © 2020 John Wiley & Sons Ltd. Mass Spec Rev

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MS Platform for Erythropoietin Glycome Characterization

Cited by 8 publications

References 44 publications

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry

NEGATIVE ION MASS SPECTROMETRY FOR THE ANALYSIS OF N‐LINKED GLYCANS

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