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 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 O126 and N83 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 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 PBA-SPE-CE-MS to better characterize N24 and N38 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.

Capillary Electrophoresis: A Three-Year Literature Review

“…The integration of phenylboronic acid as a chromatographic sorbent in an online SPE-CE-MS setup yielded a significantly improved LOD for glycopeptides in recombinant human erythropoietin digests compared to conventional methods, such as TiO 2 -based SPE. The microcartridge lifetime was also extended, making the method suitable for the analysis of glycoproteins, especially at low concentration levels …”

“…The microcartridge lifetime was also extended, making the method suitable for the analysis of glycoproteins, especially at low concentration levels. 22 Benavente's group recently demonstrated the use of an online aptamer affinity SPE-CE-MS technique (AA-SPE-CE-MS) as a promising method to improve the detection sensitivity of CE-MS for intact protein biomarkers while minimizing sample handling and increasing analytical throughput. 23−25 The utilization of functional materials, such as molecularly imprinted polymers (MIPs), 26 multiwalled carbon nanotubes, 27 metal−organic frameworks (MOFs), 28 and chemically modified natural fibers, 29 as solid-phase materials was another focus in this area.…”

Evolution of Green Sample Preparation: Fostering a Sustainable Tomorrow in Analytical Sciences

“…Table summarizes recent contributions related to green sample preparation methods for bioanalysis. ,,− As it can be seen, there is not a clear preference for the use of liquid-based or solid-based extractions. A clear trend cannot be observed in the materials/solvents selected, with both commercial and lab-made alternatives being used.…”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…Online phenylboronic acid‐based extraction of glycopeptides from enzymatic digestion of recombinant human erythropoietin increased sensitivity of CE–MS analysis of this protein 500‐times as compared to direct CE–MS and 200‐times as compared to TiO 2 –SPE–CE–MS [81]. A double‐frit particle packed FS microcartridge (250/360 µm id/od × 7 mm) filled with phenylboronic acid sorbent was inserted at 75 mm from the injection end of the CE separation capillary.…”