Sensitive Analysis of Recombinant Human Erythropoietin Glycopeptides by On-Line Phenylboronic Acid Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry.
Montserrat Mancera-ArteuFernando BenaventeVictoria Sanz-NebotEstela GiménezPublished in: Journal of proteome research (2023)
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 glycoproteins. Recombinant human erythropoietin (rhEPO) was selected as the model glycoprotein and subjected to enzymatic digestion with several proteases. The tryptic O 126 and N 83 glycopeptides from rhEPO were targeted to optimize the methodology. Under the optimized conditions, intraday precision, linearity, limits of detection (LODs), and microcartridge lifetime were evaluated, obtaining improved results compared to that from a previously reported TiO 2 -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 TiO 2 -SPE-CE-MS). Moreover, rhEPO Glu-C digests were also analyzed by PBA-SPE-CE-MS to better characterize N 24 and N 38 glycopeptides. Finally, the established method was used to analyze two rhEPO products (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 the protein digest.
Keyphrases
- mass spectrometry
- solid phase extraction
- liquid chromatography
- capillary electrophoresis
- tandem mass spectrometry
- high performance liquid chromatography
- liquid chromatography tandem mass spectrometry
- ms ms
- simultaneous determination
- high resolution mass spectrometry
- recombinant human
- molecularly imprinted
- gas chromatography
- ultra high performance liquid chromatography
- gas chromatography mass spectrometry
- multiple sclerosis
- high resolution
- energy transfer
- quantum dots
- hydrogen peroxide
- cancer therapy
- amino acid
- data analysis
- risk assessment