An MS-based Middle-Up Approach for the Allotype-Specific Analysis of Human Plasma IgG Fc N-Glycosylation Thomas Senard (Presenter) Leiden University Medical Center >> POSTER (PDF) Presenter Bio: My name is Thomas Sénard, I started my PhD one year ago in the group of Manfred Wuhrer in the Center for Proteomics and Metabolomics at Leiden University Medical Center (LUMC), The Netherlands. My PhD project is entitled “Absolute LC-MS based quantitation of serum IgG Fc N-glycosylation for patient stratification in chronic inflammatory diseases”; it is part of the GlySign - European Union’s Horizon 2020 research and innovation program under a Marie Sk³odowska-Curie grant. It is a collaboration between the LUMC and Genos Ltd, (Zagreb, HR), under the supervision of Dr. David Falck (LUMC) and Dr. Maja Puèiæ Bakoviæ (Genos). My scientific interests are focused on glycomics, mass spectrometry-based proteomics and immunology.

Authors: Thomas Sénard (1), Andrea Gargano (2,3), David Falck (1), Gestur Vidarsson (4), Manfred Wuhrer (1), Govert W. Somsen (2,3), Elena Dominguez Vega (1)
(1) Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300RC Leiden, The Netherlands, (2) Center for Analytical Sciences Amsterdam, 1098XH Amsterdam, The Netherlands, (3) Vrije Universiteit Amsterdam, 1081HV Amsterdam, The Netherlands, (4) Dept. Experimental Immunohematology, Sanquin Research, 1066CX Amsterdam, The Netherlands

Human plasma immunoglobulins G (IgG) are a complex mixture of proteins with several post- translational modifications (PTM), which are known to modulate antibody effector functions. We here propose a new middle-up strategy for the analysis of the fragment crystallizable (Fc) portion of polyclonal IgG, with a focus on specific glycosylation and PTMs of IgG allotypes. The analyses were performed by hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE) coupled with mass spectrometry (MS). These two orthogonal systems allow the resolution of the Fc allotypes with their different proteoforms, such as glycosylated, oxidized, C-terminal lysine-clipped or deamidated IgGs.

Introduction

Immunoglobulins (Ig) are glycoproteins produced by plasma cells and used by the adaptive immune system to fight pathogens. Immunoglobulin G (IgG) represents 80% of Ig in human plasma and is composed of two heavy chains and two light chains. Each heavy chain in the CH2 domain of its fragment crystallizable (Fc)-region possesses a diantennary N-linked complex glycan structure on the glycosylation site, at residue Asn297. Many past studies of plasma IgG N-glycosylation demonstrated its relevance of its impact on the immune system. For instance, galactosylation has been shown to decrease with age (2) as well as with disease activity of autoimmune diseases, including rheumatoid arthritis (RA) (1).

IgGs comprise four different subclasses and several allotypes, which are described by slight differences in the amino acid sequences of heavy or light chains of different individuals. In addition, IgGs exhibit many other post-translational modifications (PTM), such as deamidation, lysine-clipping, oxidation and glycation. These PTMs can influence protein glycosylation, and together influence the properties of IgGs including effector functions.

Current approaches to study polyclonal human IgGs glycosylation imply protein digestion (bottom-up RP-LC-MS) or glycan release (MALDI-TOF-MS). Although these approaches allow high-throughput analysis, their use inevitably brings a considerable loss of information, particularly regarding the allotypes or the interdependence of different PTMs. However, due to the complexity and inherent variability of polyclonal IgGs, their intact analysis is still not feasible.

Here we propose a middle-up strategy for analysis of Fc portions obtained from human plasma IgGs, with the aim of obtaining an integrated overview of the glycosylation and other PTMs of each specific allotype.

Methods

Polyclonal IgGs from human plasma and intravenous IgGs (IVIg), as well as monoclonal standard IgG allotypes, were captured with anti-Fc beads and digested on-bead with the bacterial protease IdeS (FabRICATOR®, Genovis). Fc portions were then eluted using formic acid and dried by vacuum centrifugation. After IgG capturing, the efficiency of digestion and separation of Fab parts and Fc portions was verified through SDS-PAGE.

Obtained intact Fc chains were analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS). CE separations were performed on a CESI 8000 instrument (Sciex), coupled to a QTOF-MS system (Bruker Impact). The separation was achieved using a polyethylenimine (PEI)-coated capillary with a background electrolytes solution of 20% acetic acid and 10% methanol.

HILIC separations were performed on a capillary-based UltiMate RSLCnano system (ThermoFisher Scientific) coupled to UHR-QTOF-MS (Bruker Maxis HD) using a Captive Spray interface from Bruker. Separations were carried out using an amide stationary phase (amideHILIC), and acetonitrile (ACN) with water gradients.

Results

The challenge of analyzing polyclonal IgGs was overcome by using a middle-up approach, in which the experimental conditions of the digestion allowed us to collect the single Fc chains of 25 kDa, containing most of the information related to the different IgG allotypes. Multiple features, such as allotypes, glycoforms or other PTMs, could be detected thanks to the orthogonality of the two MS techniques used, CE-MS and HILIC-MS.

In the CE-MS analysis, the separation is based on the charge-to-mass ratio of a molecule and is mainly driven by the amino acidic sequence. Therefore, CE provided a good separation of the different sub classes and allotypes. Moreover, detailed information about the nature of the backbone sequence of the Fc portion (e.g. C-terminal lysine-clipping and deamidation) as well as separation of charged glycoforms (e.g. sialylated species) were observed.

On the other hand, the HILIC-MS system sorts the proteoforms by hydrophilic interactions. Therefore, it was possible to resolve the different glycoforms, their oxidated variants, the C-terminal lysine-clipped and potentially deamidated forms.

Analysis of Fc portions captured from single donors showed the variability of the different allotypes within a certain population, in which each donor can have one or two allotypes for the same IgG subclass. Beyond that, the analysis of IVIg allowed to determine all the allotypes present in the Chinese and Dutch populations and showed some differences between allotypes with respect to glycosylation patterns, in particular sialylation. For the monoclonal standard IgG allotypes, the specific glycan profiles were established. Furthermore, several oxidized forms were observed for all these glycoforms.

Conclusions & Discussion

The proposed middle-up approach appears to be a promising solution in the analysis of the intact Fc part of polyclonal IgGs captured from human plasma and IVIgs. With this protocol, it was possible to identify the different proteins variants, including allotypes, glycoforms and other PTMs, and determine direct relations between them. Further studies and datasets integration are needed to confirm the findings.

The next steps in our method development will be the exploitation of MS/MS to further confirm the mass assignments, as well as the relative quantitation of different proteoforms to prove an allotype-specific glycosylation profile. Moreover, the obtained glycosylation results are currently being compared with those obtained with well-established glycopeptide protocols to support our findings. Future clinical perspectives involve the analysis of RA samples, in order to better understand the importance of the different modifications present in RA patients in comparison with healthy donors.

(1) Bondt, A., Hafkenscheid, L., Falck, D., Kuijper, T.M., Rombouts, Y., Hazes, J.M.W., Wuhrer, M., and Dolhain, R.J.E.M. (2018). ACPA IgG galactosylation associates with disease activity in pregnant patients with rheumatoid arthritis. Ann. Rheum. Dis.

(2) Parekh, R., Roitt, I., Isenberg, D., Dwek, R., and Rademacher, T. (1988). Age-related galactosylation of the N-linked oligosaccharides of human serum IgG. J. Exp. Med. 167, 1731–1736.

This project has received funding from the Glysign - European Union’s Horizon 2020 research and innovation program under the Marie Sk³odowska-Curie grant agreement No 722095.