Mass spectrometry for the identification and analysis of highly complex glycosylation of therapeutic or pathogenic proteins

Abstract

Introduction: Protein glycosylation influences characteristics such as folding, stability, protein interactions, and solubility. Therefore, glycan moieties of therapeutic proteins and proteins that are likely associated with disease pathogenesis should be analyzed in-depth, including glycan heterogeneity and modification sites. Recent advances in analytical methods and instrumentation have enabled comprehensive characterization of highly complex glycosylated proteins.

Area covered: The following aspects should be considered when analyzing glycosylated proteins: sample preparation, chromatographic separation, mass spectrometry (MS) and fragmentation methods, and bioinformatics, such as software solutions for data analyses. Notably, analysis of glycoproteins with heavily sialylated glycans or multiple glycosylation sites requires special considerations. Here, we discuss recent methodological advances in MS that provide detailed characterization of heterogeneous glycoproteins.

Expert opinion: As characterization of complex glycosylated proteins is still analytically challenging, the function or pathophysiological significance of these proteins is not fully understood. To reproducibly produce desired forms of therapeutic glycoproteins or to fully elucidate disease-specific patterns of protein glycosylation, a highly reproducible and robust analytical platform(s) should be established. In addition to advances in MS instrumentation, optimization of analytical and bioinformatics methods and utilization of glycoprotein/glycopeptide standards is desirable. Ultimately, we envision that an automated high-throughput MS analysis will provide additional power to clinical studies and precision medicine.

Keywords: N-glycosylation; O-glycosylation; Fc fusions protein therapeutics; Mucin 1 (MUC-1); erythropoietin; immunoglobulin glycosylation; intravenous immunoglobulin (IVIG); virus glycoconjugates.

Figure 1.

Biosynthesis of common N- and O-glycans. a) N-glycan synthesis pathway. The mature glycan precursor attached to dolichol phosphate (Dol-P) is transferred to the Asn residue of the Asn-X-Ser/Thr peptide sequence in the endoplasmic reticulum (ER) by oligosaccharyltransferase (OST). After cleavage of three glucose (Glc) residues and one mannose (Man) residue, properly folded glycoprotein with oligomannose N-glycan (gray shadow with one asterisk*) is transferred to the Golgi apparatus. In the Golgi apparatus, the structure of GlcNAcMan₅GlcNAc₂ is generated by further mannose removal and addition of GlcNAc. By additional trimming by α-mannosidase Ⅱ (α-Man II) and addition of second GlcNAc by GlcNAc-transferase-II (GnT-II), the precursor of all complex N-glycans is generated. Further branching and capping of antennae is performed by some transferases to synthesize complex N-glycans (gray shadow with three asterisks***): GlcNAc-transferase III-V (GnT-III-V), α1–6-fucosyltransferase (FUT8), β1,4-galactosyltransferases (Gal-T), α2,3-sialyltransferase (α2,3, Sialyl-T) and α2,6-sialyltransferase (α2,6 Sialyl-T). Hybrid N-glycans are formed if the GlcNAcMan₅GlcNAc₂ glycan is not trimmed by α-mannosidase II and then extended by Gal-T and Sialyl-T (gray shadow with two asterisks**). N-acetylglucosamine, GlcNAc; α- glucosidases, α-Glc; α-mannosidase I, α-Man I; GlcNAc-transferase I, GnT- I. b) O-glycan synthesis pathway. O-GalNAc are added to the protein in α-linkage to Ser/Thr by a polypeptide GalNAc-transferase GALNT) in the Golgi apparatus. Four core structures of O-GalNAc glycans are generated by extension of glycans by different enzymes. Less extended O-GalNAc glycans, which are linked to Ser/Thr, are called Tn antigen, T antigen, and Sialyl-Tn antigen and are associated with several types of cancer. N-acetylgalactosamine, GalNAc; galactose, Gal; N-acetylglucosamine, GlcNAc; N-acetylneuraminic acid, NeuAc; core 1 β1–3-galactosyltransferase 1, C1GALT1; molecular chaperone of C1GALT1, COSMC; core 2 β1–6-N-acetylglucosaminyltransferases 1, C2GnT-1, GCNT1; β−1,4-galactosyltransferase, B4GALT; α2–6-sialyltransferase, ST6GALNAC1; α2–3-sialyltransferases, ST3GAL1; β1–3-N-acetylglucosaminyltransferase 3, B3GNT3; core 3 β1–3 N-acetylglucosaminyltransferase 6, B3GNT6; M-type N-Acetylglucosaminyl transferase 3, GCNT3; β1,3-galactosyltransferase 5, B3GALT5.

Figure 2.

The overall strategy for the analysis of protein glycosylation. (Left) For intact glycoprotein analysis, a glycoprotein is directly subjected to high-resolution hybrid MS analysis after reduction of disulfide bonds. (Right) For glycopeptide analysis, the glycoprotein is digested with a protease(s) and the resultant glycopeptides are enriched prior to MS analysis. Glycans are released by enzymatic or chemical methods, either from glycopeptides or from glycoproteins. As shown in the lower panel, various useful technologies already exist and are rapidly advancing. peptide N-glycosidase F, PNGase-F; Hydrophilic interaction chromatography, HILIC; capillary electrophoresis, CE; differential ion mobility, DMS; collision-induced dissociation, CID; electro transfer dissociation, ETD; Quadrupole time of flight, Q-TOF.

Figure 3.

Schematic representation of IgG1 and IgA1/2 with the cleavage sites of different proteases. a) Scheme of IgG1 structure is shown in the top left. The example of enzymatic digestion for separation of IgG Fab (antigen-binding fragment) and IgG Fc (crystallizable fragment) moieties are shown in the top right. Digestion of IgG by pepsin and papain occurs below and above the disulfide bond, respectively. Conversely, IdeS and IdeZ proteases digest IgG at a specific site between Gly-Gly of the IgG hinge region, below the disulfide bond. Novel workflow of site-specific N-glycan analysis of IgG, which was reported by Bondt et al. [49], is shown in the bottom of the figure. IgG was captured and digested by IdeS protease on beads. The F(ab’)₂ fragment was collected in the flow-through (FT), and the Fc portion was collected after elution by 100 mM HCl. b) Schemes of IgA1 and IgA2 structure are shown in the top. Backbone peptide of IgA hinge region (HR) produced by trypsin is shown in the middle. O-glycosylation mainly occurs at the serine (S) and threonine (T) residues indicated in red. Digestion sites in the IgA1 HR by IgA-specific proteases (Clostridium ramosum AK183, Streptococcus pneumoniae TIGR4, Haemophilus influenzae HK50) are also shown. N-glycopeptides of IgA1/2 produced by trypsin digestion are shown at the bottom. N-glycan attachment sites are shown in red. Closed circles represent N-glycans. Open circles represent O-glycans. constant region 1, CH1; constant region 2, CH2; constant region 3, CH3; heavy-chain variable region, VH; light-chain variable region, VL

Figure 4.

Comparison of two analytical work flow for IgA-glycopeptides. a) High-throughput strategy for analysis of IgA N- and O- glycopeptides. This is the method reported by Bondt et al [102]. Purified IgA was digested by trypsin after reduction and alkylation. *To improve separation accuracy, two-step Hydrophilic interaction chromatography (HILIC) enrichment was performed, which enabled site-specific N-glycopeptide detection. The merit obtained by this method is high-throughput analysis. By using ultrahigh resolution matrix assisted laser dissociation (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS, detection accuracy was improved and sialic acid fragmentation was reduced. The representative mass spectra after two-step HILIC enrichment were shown in the middle. This MS spectrum was obtained from ref 102. under Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). O-glycopeptide, Q1; non-truncated, T1; truncated Asn340 containing glycopeptide, U1; hexose, H; N-acetylhexosamine, N; fucose, F; N-acetylneuraminic acid, S; sodium adduct, *; unidentified non-IgA-related glycopeptide contaminant, # b) In-depth IgA O-glycopeptide analysis. Purified IgA1 was digested by neuraminidase and trypsin. By using an online nano liquid chromatography (LC) system, glycopeptide enrichment is not necessary before measurement by MS. Representative mass spectrum is shown in the middle. LC system coupled to MS enabled differentiation between isomeric forms. By shortening the hinge-region (HR) peptide with IgA-specific protease, detection of the attachment sites was demonstrated by Takahashi et al. using LC-electron transfer dissociation (ETD)-MS/MS [111].

Figure 5.

The workflow for Etanercept N- and O-glycosylation analysis. a) The analysis workflow for O-glycosylation of Etanercept. a-1) By native MS analysis after peptide N-glycosidase F (PNGase-F) digestion, the number of O-glycan core variants with different numbers of sialic acids was detected, and the ratio of NeuAc residues to O-glycan core was elucidated. a-2) After PNGase-F and sialidase digestion, the number of O-glycans was detected by native MS. a-3) attachment sites of O-glycans were analyzed by electron transfer dissociation (ETD) fragmentation after digestion by trypsin and sialidase. N-acetylneuraminic acid, NeuAc b) The analysis workflow of N-glycosylation of Etanercept. b-1) By IdeS digestion, followed by sialidase and O-glycosidase digestion, the N-glycoform of the tumor necrosis factor-α receptor (TNFR) domain was elucidated using native MS. b-2) After IdeS digestion, the N-glycoform of the Fc domain was elucidated using native MS. b-3) To pinpoint the exact N-glycan structures on Asn¹⁴⁹ and Asn¹⁷¹, glycopeptides digested by AspN were analyzed by HPLC-MS/MS. The N-glycan structures of Asn³¹⁷ were elucidated by trypsin-digested glycopeptide analysis. endoproteinase AspN, AspN