Strategies for Glycoprotein Analysis

Understanding the Importance of Glycosylation

  • Fig. 1: Separation of isomeric N-glycans on a carbon (PGC) column with detection by electrospray mass spectrometry (ESI-MS). The extract mass trace for the composition 5 hexoses + 4 N-acetylglucosamines (GlcNAc) + 1 fucose is shown. The hexoses are mannose and/or galactose.Fig. 1: Separation of isomeric N-glycans on a carbon (PGC) column with detection by electrospray mass spectrometry (ESI-MS). The extract mass trace for the composition 5 hexoses + 4 N-acetylglucosamines (GlcNAc) + 1 fucose is shown. The hexoses are mannose and/or galactose.
  • Fig. 1: Separation of isomeric N-glycans on a carbon (PGC) column with detection by electrospray mass spectrometry (ESI-MS). The extract mass trace for the composition 5 hexoses + 4 N-acetylglucosamines (GlcNAc) + 1 fucose is shown. The hexoses are mannose and/or galactose.
  • Fig. 2: Glycopeptide spectrum of mouse IgG1. The figure shows a sum spectrum of a section of the reversed-phase chromatogram acquired by ESI-MS. Peaks are triply charged ions of the same peptide with glycans of different size.
  • Fig. 3: Deconvoluted ESI-MS spectrum of an IgG antibody. The 162 Da increments caused by the increasing number of galactose residues are evident.

Most modern biopharmaceuticals just as most serum proteins consist of more than amino acids. They carry complex sugar chains that exert – e.g. in immunology and oncology - a variety of still only partially understood functions. Glycoprotein analysis is of utmost importance in “red” biotechnology, where serum glycoproteins such as antibodies (IgG), interferons, interleukins, growth factors such as erythropoietin (EPO) or hormones such as follitropin are produced using mammalian cells in huge fermenters. Glycosylation modifies their efficacy and biological half-life in the patient´s body and it even effect adverse side reactions. The thus mandatory control of glycosylation may be realized by three strategies: a) analysis of glycans released from the protein; b) glycopeptide analysis and c) analysis of the intact (glyco-)protein.

Analysis of Free Glycans

At first, the sugar chains are released from the protein. In the case of the asparagine-linked N-glycans this can be done enzymatically with PNGase F. The now free glycan has a reducing end to which fluorogenic reagents can be specifically attached. The serine or threonine bound O-glycans are released by base in the presence of NaBH4 as reduced glycans. Should a reducing end be desired, one has to resort to the somewhat unpopular anhydrous hydrazine.

Analysis of reducing N- or O-glycans is mostly accomplished after labeling with fluorogenic amine such as 2-aminobenzamide on hydrophilic columns. This hydrophilic liquid interaction chromatography (HILIC) separates primarily according to size.

For samples of moderate complexity such as IgG this constitutes a next to perfect method. For more complex glycans even the current sub 2 µm UPLC columns reach their separation limits. Coupling to an electrospray mass analyzer works well with procaine-labeled glycans, at least for not too large ones.

For highly complex mixtures, mass spectrometry (MS) with its much higher resolution lends itself as the method of choice. MALDI-TOF MS offers ease of use and high speed. As hexoses, pentoses, deoxyhexoses, N-acetylhexosamines and sialic acids possess different masses, the composition of glycans is readily deduced from their mass.

However, isobars such as mannose or galactose are not discriminated and the isomeric structure of glycans remains hidden. The simple and abundant serum glycan Hex5HexNAc4Fuc for example can occur in over thirty isomeric forms. Structural informations are obtained through fragmentation, which in MALDI-TOF MS is induced by increased laser energy. The labile sialic acids have to be derivatized [3]. In case of permethylation, all OH-groups are converted to methyl ethers. This extra effort is rewarded by information-rich fragment spectra.

The most elaborate approach is application of LC-ESI-MS, for which reversed-phase columns cannot be used as glycans don’t adhere to them. The somewhat delicate „porous graphitic carbon“ (PGC) can bind free sugars and exhibit high shape selectivity, i.e. they separate isomeric glycans very well [4]. Thus, also α and β forms of reducing glycans are separated and hence the sugars are reduced to obtain only one peak. The method is therefore a priori also suitable for reduced O-glycans. The drawback of peaks hiding in a huge amount of data and becoming visible only through „extracted ion chromatograms“ (EICs) is rewarded by the ability to separate isomers, which can then be assigned either by their elution position and/or fragment ion spectrum (CID-MS/MS). The example demonstrates the separation of isobaric glycans of the composition Hex5HexNAc4Fuc on a PGC-column (fig. 1).

While for most analytes, positive-ion mode is the choice, glycans allow both polarities. In fact, even highly sialylated glycans ionize well in positive mode and neutral glycans yield negative ions with surprising efficiency. The fragment spectra obtained from positive ions are easy to interpret but lack hints for assignment of the isomeric form. They also tend to intramolecular re-arrangements unless they are alkali adduct ions. Negative ion spectra convince due to the occurrence of ring cleavages, which allow valuable insights about structural details (fig. 1). Determination of molar ratios, however, is difficult for glycans differing in size and charge [5].

Analysis of Glycopeptides

To obtain suitably small pieces, the glycoprotein is digested with a protease – usually trypsin. As in standard proteomics the peptides and now also glycopeptides are subjected to reversed-phase chromatography coupled to ESI-MS. Retention is dominated by the peptide moiety. Only sialylated glycopeptides are additionally retained by the anion exchange properties of most reversed-phase columns. Choice of a solvent with ionic strength makes all glycoforms of a given peptide elute within a very narrow time window (fig. 2). Often, a fraction of the (glyco-)peptide occurs in unglycosylated form, which elutes 1-2 min later. In large data sets, glycopeptides can be bound via characteristic B-fragment ions, their calculated total mass (peptide plus possible glycan masses) or subtle software approaches [6, 7].

Ionization and detection efficiency are essentially governed by the peptide part und thus molar proportions of glycoforms including the “site occupancy” can be deduced from peak heights. High resolution instruments such as Q-TOF or Orbitrap mass spectrometers are recommended tools.

Intact Mass Analysis

This so called “top down” approach benefits from the tendency of large molecules to acquire a likewise large number of charges. Thus antibodies and even larger proteins become accessible to Q-TOF or Orbitrap mass spectrometers. Effective desalting is mandatory and is often accomplished on-line with wide-bore C4- or C8-silica or hydrophobic monolith columns [8, 9]. The glycoforms of antibodies can readily be seen (fig. 3). Intact mass analysis measures the sum of all modifications that occurred to the protein including lysine clipping (in IgG), disulfide-bridge and pyroglutamate formation, acylation and others. Interpretation of such spectra is therefore often very difficult and relies on information from the types of analyses described above. Some promise lies in direct fragmentation of protein ions [10].


Friedrich Altmann

Prof. Dr. Dipl.-Ing. Friedrich Altmann

University of Natural Resources and Life Sciences, Vienna
Department of Chemistry
Vienna, Austria


More on mass spectrometry


[1] J. Ahn et al., Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent, J Chromatogr B Analyt Technol Biomed Life Sci 878 (2010) 403. DOI: 10.1016/j.jchromb.2009.12.013

[2] M.A. Lauber et al., Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection, Anal Chem 87 (2015) 5401. DOI: 10.1021/acs.analchem.5b00758

[3] N. de Haan et al., Sialic Acid Derivatization for the Rapid Subclass- and Sialic Acid Linkage-Specific MALDI-TOF-MS Analysis of IgG Fc-Glycopeptides, Methods Mol Biol 1503 (2017) 49. DOI: 10.1007/978-1-4939-6493-2_5

[4] M. Pabst, F.  Altmann, Glycan analysis by modern instrumental methods, Proteomics 11 (2011) 631. DOI: 10.1002/pmic.201000517

[5] C. Grunwald-Gruber et al., Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry, Anal Bioanal Chem 409 (2017) 2519. DOI: 10.1007/s00216-017-0235-8

[6] M. Pabst et al., Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140, Biol Chem 393 (2012) 719. DOI: 10.1515/hsz-2012-0148

[7] J. Stadlmann et al., Comparative glycoproteomics of stem cells identifies new players in ricin toxicity, Nature 549 (2017) 538. DOI: 10.1038/nature24015

[8] J. Mohr et al., High-efficiency nano- and micro-HPLC--high-resolution Orbitrap-MS platform for top-down proteomics, Proteomics 10 (2010) 3598. DOI: 10.1002/pmic.201000341

[9] T. Wohlschlager et al., Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals. Nat Commun 9 (2018), 1713. DOI:  10.1038/s41467-018-04061-7

[10] T.P. Cleland et al., High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer, J Proteome Res 16 (2017) 2072. DOI: 10.1021/acs.jproteome.7b00043



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