N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

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 samples should be vortexed until all solid proteins are dissolved. Human plasma (lyophilized) was used for the preparation of the plasma samples.
   ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Carlo Erba Reagents	401413	Glacial RS For LC/MS
Acetonitrile	Merck	1000292500	LC-MS LiChrosolv
Agilent 1200 Series HPLC with 1260 Series FLD dedector	Agilent Technologies
Ammoniumm Formate	Carlo Erba Reagents	419741	For LC/MS
Bruker TIMS-TOF (Q-TOF) Mass Spectrometry	Bruker Daltonics
Cellulose	Sigma Aldrich	310697	microcrystalline, powder, 20 μm
Deionized Water	Carlo Erba Reagents	412111	For LC/MS
Dimethyl sulfoxide	Sigma Aldrich	41639	BioUltra, for molecular biology, ≥99.5% (GC)
Empty polypropylene SPE Tube with PE frits	Sigma Aldrich	54220	20 μm porosity,volume 1 mL
Extraction Manifold, 20-position	Waters	WAT200607	Complete with rack for 13 x 100 mm tubes
Human Plasma	Sigma Aldrich	P9523	lyophilized
IGEPAL CA-630	Sigma Aldrich	I8896	for molecular biology
IgG	Sigma Aldrich	I4506	lyophilized powder
Phosphate buffered saline	Sigma Aldrich	P4417	Tablet
PNGase F enzyme	Promega	V483A
Procainamide hydrochloride	abcam	ab120955
Sodium cyanoborohydride	Sigma Aldrich	156159	reagent grade, 95%
Sodium dodecyl sulfate	Sigma Aldrich	71725
trastuzumab	Roche Diagnostics
Trifluoroacetic acid	Sigma Aldrich	302031	for HPLC, ≥99.9%