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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Abstract

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

Introduction

Glycosylation is a vital post-translational modification observed in proteins1. Multiple enzymatical processes regulate glycosylation modification in cellular organisms. Glycans are attached to the proteins by these enzymatical processes, and the proteins subjected to this modification are called glycoproteins1. Two glycosylation types are commonly observed in proteins. O-glycosylation is the attachment of O-glycans to the side chain of serine or threonine amino acid residues. N-glycosylation is the attachment of N-glycans to the side chain of asparagine amino acid residue in a protein....

Protocol

NOTE: The human plasma used is commercially available (Table of Materials). No further biological samples obtained from humans were used.

1. ​Glycan release

  1. Denaturation of (glyco-)proteins
    1. Prepare the glycoprotein standards (e.g., IgG, a monoclonal antibody) at a concentration of a 10 µg·µL-1 in deionized H2O. For human plasma, the concentration used is 70 µg·µL-1.
      NOTE: The s.......

Representative Results

In this presented approach, the N-glycans were first released, labeled by the procainamide tag and purified by cellulose-containing SPE cartridges. Then, N-glycan analysis of IgG, trastuzumab, and human plasma were performed by an HPLC-HILIC-FLD-MS/MS system. The MS (base peak) and FLD chromatograms of the determined N-glycan structures obtained from IgG and trastuzumab are shown in Figure 1, respectively. The MS/MS data obtained from these analyses were imported t.......

Discussion

N-glycan profiling of glycoproteins includes challenging steps. Although there are many different methodologies for this purpose, a suitable approach should be selected for both identification and quantification of N-glycan structures14. HILIC-FLD is the gold standard approach for the quantification of N-glycans. However, identification of all N-glycan types by FLD detection is not achieved. Therefore, tandem MS analysis is needed for confirming N-glyca.......

Acknowledgements

This work was partly supported by the Ministry of Development-Republic of Turkey with project number: 2016 K121230. Bekir Salih gratefully acknowledges the Turkish Academy of Science (TUBA) for the partial financial support.

....

Materials

NameCompanyCatalog NumberComments
Acetic acidCarlo Erba Reagents401413Glacial RS For LC/MS
AcetonitrileMerck1000292500LC-MS LiChrosolv
Agilent 1200 Series HPLC with 1260 Series FLD dedectorAgilent Technologies
Ammoniumm FormateCarlo Erba Reagents419741For LC/MS
Bruker TIMS-TOF (Q-TOF) Mass SpectrometryBruker Daltonics
CelluloseSigma Aldrich310697microcrystalline, powder, 20 μm
Deionized WaterCarlo Erba Reagents412111For LC/MS
Dimethyl sulfoxideSigma Aldrich41639BioUltra, for molecular biology, ≥99.5% (GC)
Empty polypropylene SPE Tube with PE fritsSigma Aldrich5422020 μm porosity,volume 1 mL
Extraction Manifold, 20-positionWatersWAT200607Complete with rack for 13 x 100 mm tubes
Human PlasmaSigma AldrichP9523lyophilized
IGEPAL CA-630Sigma AldrichI8896for molecular biology
IgGSigma AldrichI4506lyophilized powder
Phosphate buffered salineSigma AldrichP4417Tablet
PNGase F enzymePromegaV483A
Procainamide hydrochlorideabcamab120955
Sodium cyanoborohydrideSigma Aldrich156159reagent grade, 95%
Sodium dodecyl sulfateSigma Aldrich71725
trastuzumabRoche Diagnostics
Trifluoroacetic acidSigma Aldrich302031for HPLC, ≥99.9%

References

  1. Dwek, R. A. Glycobiology: Toward understanding the function of sugars. Chemical Reviews. 96 (2), 683-720 (1996).
  2. Varki, A. Biological roles of glycans. Glycobiology. 27 (1), 3-49 (2016).
  3. Spiro, R. G.

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N glycan ProfilingGlycoproteinsHydrophilic Interaction Liquid ChromatographyFluorescence DetectionMass SpectrometryProcainamide LabelingBiomarker DiscoveryGlycosylation PatternsBiopharmaceutical ProteinsGlycan Characterization

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