Dataset Information

SENSITIVE ANALYSIS OF RECOMBINANT HUMAN ERYTHROPOIETIN GLYCOPEPTIDES BY ON-LINE PHENYLBORONIC ACID SOLID-PHASE EXTRACTION CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

ABSTRACT: In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl-hydrophilic interaction (aminopropyl-HILIC) and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS). As PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O126 and N83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, repeatabilityintraday precision, linearity, limits of detection (LODs) and microcartridge lifetime were evaluated, obtaining improved results compared to a previously reported TiO2-SPE-CE-MS method, especially for LODs of N-glycopeptides (up to 500 times lower than by CE-MS and up to 200 times lower than by TiO2-SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by Finally, the PBA-SPE-CE-MS method was applied to the analysis of rhEPO glycopeptides from Glu-C digests to better characterize N24 and N38 glycopeptidessites. Finally, the established method was used to analyze two rhEPO biosimilars (EPOCIM and NeuroEPO plus), demonstrating its applicability in biopharmaceutical analysis. The sensitivity of the proposed PBA-SPE-CE-MS method improves the existing CE-MS methodologies for glycopeptide analysis and shows a great potential in glycoprotein analysis to deeply characterize protein glycosites even at low concentrations of protein digest.

INSTRUMENT(S): 6220 Time-of-Flight LC/MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Montserrat Mancera Arteu

LAB HEAD: Estela Giménez López

PROVIDER: PXD039282 | Pride | 2023-02-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CE_MS_1000ppm_EPO_CRS.mzML	Mzml
	Glycopeptides_EPO_neuroEPO__SEARCH_.xlsx	Xlsx
	SPE_CE_MS_50ppm_EPO_CIM.mzML	Mzml
	SPE_CE_MS_50ppm_EPO_CRS.mzML	Mzml
	SPE_CE_MS_50ppm_EPO_CRS_GluC.mzML	Mzml

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Publications

Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.

Mancera-Arteu Montserrat M Benavente Fernando F Sanz-Nebot Victoria V Giménez Estela E

Journal of proteome research 20230210 3

In this study, several chromatographic sorbents: porous graphitic carbon (PGC), aminopropyl hydrophilic interaction (aminopropyl-HILIC), and phenylboronic acid (PBA) were assessed for the analysis of glycopeptides by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS). As the PBA sorbent provided the most promising results, a PBA-SPE-CE-MS method was developed for the selective and sensitive preconcentration of glycopeptides from enzymatic digests of glycoprote ...[more]

PMID: 36763563

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