State-of-the-art Glycopeptide Identification Using an Orbitrap Astral Mass Spectrometer

Importance of the topic

Protein glycosylation is a key post-translational modification that modulates protein folding, stability, and cellular signaling. Reliable identification and site localization of glycopeptides are critical for understanding disease mechanisms, discovering biomarkers, and developing biotherapeutics. However, the complexity and dynamic range of clinical samples such as human plasma present significant analytical challenges.

Objectives and overview

This study aimed to demonstrate the capabilities of the Thermo Scientific Orbitrap Astral mass spectrometer for deep glycopeptide profiling. By combining high-performance chromatography with data-dependent acquisition (DDA) employing stepped collision energies, the authors evaluated speed, sensitivity, and identification confidence in enriched human plasma samples.

Methodology and instrumentation

Sample preparation:

• 400 µL human plasma depleted of abundant proteins (HSA/IgG resin)
• Proteolytic digestion using EasyPep Maxi kit
• Glycopeptide enrichment via Oasis MAX SPE in HILIC mode

Chromatography and mass spectrometry:

• Vanquish Neo UHPLC with Aurora Ultimate TS C18 column (25 × 75 mm, 1.6 µm)
• Mobile phases: A = water + 0.1% FA, B = 80% ACN + 0.1% FA
• Orbitrap Astral MS in positive-ion DDA mode
• Stepped collision energies (two and three NCE steps per scan) implemented via IRM accumulation and parallel Astral analysis

Data analysis:

• PMI-Byonic v5.3.44 search against UniProt human database and glycan libraries
• Parameters: 10 ppm precursor, 20 ppm fragment tolerance, 1% protein FDR, Byonic score > 200

Main results and discussion

The Orbitrap Astral MS achieved scan rates up to 200 Hz, representing up to 3× faster DDA acquisition compared to the Orbitrap Exploris 480. Using stepped collision energies increased the number of unique glycopeptide identifications by approximately 33% (over 3 000 glycopeptides), and improved fragmentation quality as reflected by higher Byonic scores. Parallelization of ion accumulation and detection enabled continuous duty cycles and enhanced throughput.

Benefits and practical applications

The accelerated acquisition and improved confidence in glycopeptide identification facilitate comprehensive glycoproteomic analyses of complex biological samples. High-throughput workflows can support clinical biomarker discovery, QA/QC in biopharmaceutical development, and detailed structural elucidation of glycan heterogeneity.

Future trends and potential applications

Advances may include integration of data-independent acquisition with stepped energies, hybrid fragmentation strategies (HCD + ETD/EThcD), and real-time search algorithms. Broader application to other PTMs and large-scale clinical cohorts will further expand the impact of high-speed, high-sensitivity mass spectrometry.

Conclusion

The Thermo Scientific Orbitrap Astral mass spectrometer, combined with stepped collision energy DDA, delivers rapid, sensitive, and high-confidence glycopeptide profiling in human plasma. This platform represents a significant step forward for large-scale glycoproteomics and translational research.

Reference

• Baerenfaenger M., et al. Maximizing Glycoproteomics Results through an Integrated Parallel Accumulation Serial Fragmentation Workflow. Analytical Chemistry, 96(22), 8956–8964 (2024).
• Stewart H., et al. Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis. Analytical Chemistry, 95(42), 15656–15664 (2023).