Native Top‐Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies

Abstract Oligomerization and glycosylation modulate therapeutic glycoprotein stability and efficacy. The interplay between these two critical attributes on therapeutic glycoproteins, is however often hard to define. Here, we present a native top‐down mass spectrometry (MS) approach to assess the glycosylation status of therapeutic cytokine and hormone assemblies and relate interfacial glycan occupancy to complex stability. We found that interfacial O‐glycan stabilizes tumor necrosis factor‐α trimer. On the contrary, interferon‐β1a dimerization is independent of glycosylation. Moreover, we discovered a unique distribution of N‐glycans on the follicle‐stimulating hormone α subunit. We found that the interfacial N‐glycan, at Asn52 of the α subunit, interacts extensively with the β subunit to regulate the dimer assembly. Overall, we have exemplified a method to link glycosylation with assembly status, for cytokines and hormones, critical for informing optimal stability and bioavailability.

were analysed on a Dionex Ultimate 3000 UHPLC coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The peptides were firstly loaded onto a 75 μm×2 cm precolumn and separated on a 75 μm×15 cm Pepmap C18 analytical column (Thermo Fisher Scientific) with a binary buffer system. Buffer A was 0.1% formic acid (FA) in 100% H2O and buffer B was 0.1% FA in 80% acetonitrile with 20% H2O. The Eclipse mass spectrometer was operated in data-dependant acquisition mode with one full MS scan followed by MS/MS scans with HCD fragmentation.
Denaturing MS analysis. The glycoproteins were diluted to 1% formic acid for denaturing. The denatured glycoproteins were then analysed on a Dionex Ultimate 3000 UHPLC coupled to an Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The glycoproteins were directly loaded onto a 75 μm×15 cm Pepmap C8 analytical column (Thermo Fisher Scientific) with a binary buffer system. Buffer A was 0.1% formic acid (FA) in 100% H2O and buffer B was 0.1% FA in 80% acetonitrile with 20% H2O. The Orbitrap XL mass spectrometer was operated in MS scan mode. The denaturing mass spectra were analysed using UniDec software. The fragment ions were detected in the Orbitrap. The top-down mass spectra were analysed manually for N-glycan assignments.
Native MS data analysis. The native mass spectra were deconvoluted using UniDec software 2 . The theoretical molecular weights of IFN-β1a, TNF-α and FSH were calculated using amino acid and monosaccharide residue masses. The quantification of each proteoform was manually performed using Xcalibur.
Proteomics data analysis. The LC-MS/MS data were processed with PGlyco (version 2.0) 3 for glycopeptide identification. Quantification of the site-specific microheterogeneity was performed manually using Xcalibur (version 4.1, Thermo Fisher Scientific). The extracted ion chromatogram (XIC) of each glycopeptide was processed with 50 ppm mass tolerance and a 7-point Gaussian smoothing. The area under the curve (AUC) was integrated for glycopeptide quantification.
Denaturing MS data analysis. The mass spectra of denatured glycoprotein subunits were retrieved from LC-MS raw data using Xcalibur, and then deconvoluted using UniDec software.
Prediction of glycoprotein dimer proteoforms. The theoretical glycoprotein dimer proteoforms were calculated based on the hypothesis that the glycoprotein dimerization is independent of the glycosylation status of the monomer. The mass of the dimer proteoform D ( ) can be calculated as:

= +
whereas and are the masses of the monomer proteoforms i and j.
The corresponding relative abundance of the dimer proteoform D ( ) can be calculated as:

= ×
whereas and are the normalized relative abundances of the monomer proteoforms i and j.
The data were processed and plotted with seaborn library in Python 3.8.5. The Pearson correlation efficiency between the predicted and native MS measured datasets was calculated using pearsonr function in SciPy library.