Comprehensive Analysis of IgE Glycoforms by FAIMS-LC-MS/MS using Orbitrap Eclipse Mass Spectrometer

Importance of the Topic

Glycosylation of immunoglobulin E (IgE) is a key mediator of allergic inflammation and anaphylaxis. Detailed mapping of N-linked glycosylation sites and their microheterogeneous glycoforms is essential for understanding IgE function, improving biopharmaceutical quality control and advancing clinical glycoproteomics. However, the inherent complexity of glycopeptides and their low abundance in biological samples present significant analytical challenges.

Objectives and Study Overview

This study aimed to comprehensively identify all seven N-linked glycosylation sites on human IgE and profile their attached glycoforms. By integrating High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS Pro) with high-resolution LC-MS/MS on an Orbitrap Eclipse mass spectrometer, the workflow was optimized to enhance glycopeptide separation, signal-to-noise and dynamic range without requiring prior enrichment or fractionation.

Methods and Applied Instrumentation

Sample Preparation and Digestion

• Recombinant human IgE was reduced, alkylated and subjected to LysC/trypsin or chymotrypsin digestion to generate peptides amenable to both HCD and ETD fragmentation.

Liquid Chromatography

• Thermo Scientific EASY-nLC 1200 UPLC system with an EASY-Spray 50 cm × 75 µm column operated over a 120 min gradient (5–40 % B followed by 40–98 % B) at 300 nL/min.

Mass Spectrometry and FAIMS

• Orbitrap Eclipse Tribrid mass spectrometer equipped with FAIMS Pro interface.
• FAIMS compensation voltages (CV) from –30 V to –60 V were tested using external stepping and internal CV-stepping modes; dual CV (–45/–60 V) was highlighted.
• Data-dependent acquisition combining HCD, EThcD or HCD-pd-EThcD: MS1 at 60–120 K resolution, MS2 at 30–60 K resolution.

Data Analysis

• Proteome Discoverer 2.4 with Byonic node and Byologic, Thermo Scientific BioPharma Finder 4.0.
• Search against UniProt IgE sequence plus contaminants and a library of 57 human plasma glycans; 1 % FDR at peptide spectral match level; manual validation of glycosite assignments.

Results and Discussion

The FAIMS-LC-MS/MS workflow significantly outperformed conventional LC-MS/MS by increasing the number of glycopeptide spectrum matches and unique glycoform identifications across all seven N-linked sites. Internal CV-stepping, particularly at –45/–60 V, yielded the highest coverage for partially glycosylated sites such as N383 and the biologically critical N394 site. FAIMS improved signal-to-noise and dynamic range, facilitating detection of low-abundance complex biantennary and oligomannose glycoforms.

• HCD fragmentation provided characteristic glycosidic cleavages while ETD delivered robust peptide backbone sequencing, enabling unambiguous localization of each glycosylation site.
• All seven N-linked sites (N140, N168, N218, N265, N371, N383, N394) were confirmed in both glycosylated and nonglycosylated forms.
• Dual CV method (–45/–60 V) outperformed single-CV approaches and showed minimal sialic acid loss compared to CVs below –60 V.

Benefits and Practical Applications of the Method

This integrated FAIMS-LC-MS/MS strategy enables comprehensive glycoform profiling of heavily glycosylated proteins without extensive sample cleanup. It enhances throughput and sensitivity in biopharmaceutical development, quality control of therapeutic antibodies, and clinical biomarker discovery by resolving microheterogeneous glycopeptides more effectively.

Future Trends and Opportunities

• Integration of complementary ion mobility techniques to resolve glycan isomers and further improve separation.
• Enhanced fragmentation methods and acquisition strategies to detect ultra-low abundance glycoforms.
• Machine learning–based algorithms for automated glycopeptide identification, quantification and site localization.
• Application to other complex biologics and immunoglobulin subclasses for broader clinical and industrial impact.

Conclusion

Coupling FAIMS Pro with LC-MS/MS on an Orbitrap Eclipse provides a powerful platform for in-depth analysis of IgE glycosylation. The optimized workflow, particularly internal CV-stepping at –45/–60 V and combined HCD/ETD fragmentation, delivers enhanced glycoform coverage and confidence in site assignments, offering valuable insights for allergy research and biotherapeutic characterization.

References

• Hebert AS, Prasad S, Belford MW, et al. Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer. Anal Chem. 2018;90(15):9529–9537.
• Pfammatter S, Bonneil E, McManus FP, et al. A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Measurements. Mol Cell Proteomics. 2018;17(10):2051–2067.
• Alving K, Alving A, Wang B. Determination of Human Immunoglobulin Glycoforms by timsTOF Pro Sequencing Analysis. ASMS 2019, WP 348.
• Wu G, Hitchen PG, Panico M, North SJ, et al. Glycoproteomic Studies of IgE from a Novel Hyper IgE Syndrome Linked to PGM3 Mutation. Glycoconj J. 2016;33(3):447–456.
• Shade KC, et al. A Single Glycan on IgE Is Indispensable for Initiation of Anaphylaxis. J Exp Med. 2015;212(4):457–467.
• Plomp R, et al. Recent Advances in Clinical Glycoproteomics of Immunoglobulins. Mol Cell Proteomics. 2016;15(6):2217–2228.