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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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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Project description:Recent advances in software-driven glycopeptide identification in LC-MS/MS-based N-glycoproteomics have facilitated biochemical studies reporting thousands of intact N-glycopeptides, i.e. N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we explore the efficiency of site-specific glycoprofiling using the PTM-centric search-engine Byonic relative to manual expert annotation. To allow an appropriately deep comparison, the study utilised typical glycoproteomics acquisition and data analysis strategies, but of a single glycoprotein, the uncharacterised N-glycosylated (Asn160, Asn268 and Asn302) human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS acquisition of tryptic N-glycopeptides and released N-glycans. The basigin N-glycosylation sites, which showed extensive micro- and macro-heterogeneity, were then glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS data. The glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target and decoy proteome databases. The search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided very high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the glycoprofiling efficiency and analysis time. Automated identification of peptide modifications (methionine oxidation/carbamidomethylation) that coincide with monosaccharide mass differences (Fuc/Hex/HexNAc) and accurately distinguishing isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compositions remain challenging, arguing particular attention to such “difficult-to-identify” N-glycopeptides. The presented analysis provides valuable insights into automated glycopeptide identification; knowledge that facilitates further developments in FDR-based glycoproteomics.

Project description:Changes in IgG glycosylation have been reported to be associated with colorectal cancer (CRC), but the alteration in specific subclass of IgG is unknown. Herein, we optimized five common IgG glycopeptides enrichment methods to acquire comprehensive profiling of IgG glycoproteomics in CRC. Notably, incomplete tryptic digestion of IgG occurred when using ordinary ratio of protease to protein, which significantly impacted the final statistical analysis of association between IgG glycosylation and CRC. We introduced a method based on two-step enzymatic digestion, enabling the complete digestion of IgG glycopeptides and further improving the detection intensity of the target glycopeptides. By this approach, total 47 IgG glycopeptides can be identified by nanoLC-ESI-MS/MS. Following rapid and automatic data processing through the self-developed program, we observed that IgG1_H3N4F1 and IgG1_H3N4 were substantially increased in CRC, while IgG1_H5N5F1, IgG1_H5N4F1S1and IgG2_H5N4F1 were markedly decreased. Strikingly, these four glycopeptides were continually significantly changed in early and late stages of CRC. Further evaluation of the diagnostic performance showed they all obtained a fair performance in discriminating the patients from the normal. In terms of the glycan features, it demonstrated that CRC progression associates with the increased agalactosylation, and the decreased digalactosylation and galactosylation per antenna on diantennay glycans of IgG1 and IgG2. Concurrently, the decreased sialylation of IgG1 was strongly correlated with CRC. Moreover, tumor-specific glycosylation analysis showed that the alterations of subclass-specific IgG glycosylation were more significant in colon cancer, and no obvious difference between colon and rectal cancer was found. More importantly, all these findings were validated in an independent cohort. This two-step enzymatic approach is crucial to explore the relationship between the disease progression and IgG glycosylation.

Dataset Information

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

Publications

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

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