One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography-(ultra-)high-performance liquid chromatography [HILIC-(U)HPLC]. Here, we compare the data obtained by (U)HPLC-fluorescence (FLR) coupled to electrospray ionization-mass spectrometry (ESI-MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.
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http://dx.doi.org/10.1016/j.ab.2015.06.006 | DOI Listing |
Sci Rep
November 2024
Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Republic of Singapore.
The increasing demand for biotherapeutics has necessitated the evaluation of their critical quality attributes, one of which is glycosylation, an essential post-translational modification found on many biological molecules. In particular, the purification of N-glycans after their release from the proteins and derivatization is important in ensuring the removal of the deglycosylated protein, excess labelling reagents and salts for subsequent analysis. However, current methods of N-glycans purification are either expensive, laborious, time-consuming or not catered for high throughput analysis.
View Article and Find Full Text PDFBiomolecules
January 2024
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO -glycosylation influences important pharmacological parameters, prominently serum half-life.
View Article and Find Full Text PDFJ Chromatogr B Analyt Technol Biomed Life Sci
January 2023
Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea; Department of Global Innovative Drugs, Graduate School of Chung-Ang University, 84, Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea. Electronic address:
Background: N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods.
Objective: To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins.
Methods: N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)].
Anal Biochem
June 2022
Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06974, Republic of Korea; Department of Global Innovative Drugs, Graduate School of Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06974, Republic of Korea. Electronic address:
Sialylated and core-fucosylated N-glycans in human transferrin (HTF) are used as glycan biomarkers due to their increased or decreased characteristics in certain diseases. However, their absolute quantities remain unclear. In this study, N-glycans of HTF were identified by UPLC and LC-MS/MS using fluorescence tags [2-aminobenzamide (AB) and procainamide (ProA)] and columns [HILIC and anion exchange chromatography-HILIC (AXH)].
View Article and Find Full Text PDFAnal Chem
March 2022
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured.
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