High-throughput Glycoproteomics: deciphering age-related Glycosylation changes in the mouse brain

Glycoproteomics is essential for studying diseases like cancer, cardiovascular disorders, autoimmune diseases, neurodegenerative conditions, and metabolic syndromes. However, its progress is limited by the lack of sensitive, high-throughput technologies. We introduce a novel glycoproteomics sample preparation pipeline, sLWAC-HTP, capable of processing up to 400 samples within a 3-5 hour timeframe. Utilizing the Orbitrap Astral mass spectrometer, known for its exceptional speed and sensitivity, our comprehensive high-throughput glycoproteomics platform integrates sLWAC-HTP for sample preparation, Orbitrap Astral MS for analysis, and GlycoDIA for data processing.

This platform enabled us to investigate age-related glycosylation alterations in distinct compartments of the mouse brain, providing insights into glycosylation dynamics during brain development, maturation, and aging. Brain regions from male C57BL/6J mice at various ages—postnatal days 14 and 21, and at 2, 5, 18, and 20 months—were dissected, snap-frozen, and stored at -80°C. Tryptic O-glycopeptides from different brain compartments across six age points, each with four biological replicates, were enriched using the Jacalin lectin-based sLWAC-HTP platform. Peptides were separated by reversed-phase UHPLC on a PepMap 150 μm x 15 cm column over a 3.1-minute linear gradient using the Thermo Scientific Vanquish Neo system in Trap&Elute mode. Data were acquired on a Thermo Scientific Orbitrap Astral mass spectrometer in high-speed narrow window data-independent acquisition (DIA) mode and processed using Spectronaut software.

Our miniaturized lectin-based platform, evolved from the standard sLWAC method, has advanced to a high-throughput variant termed sLWAC-HTP. This innovation enables rapid glycopeptide enrichment from complex proteomic samples with enhanced efficiency, comparable to the traditional LWAC method. The system has been optimized for high-throughput applications using a 96-well plate format and a standard benchtop centrifuge, allowing processing up to 4x96 samples in approximately 3-5 hours. Using the Orbitrap Astral MS with an ultra-short 4-minute LC gradient and the GlycoDIA platform, we achieved a throughput of 220 samples per day.

Applying this pipeline for quantitative glycoproteomics analysis of 10 different brain compartments at six age points, we detected, on average, 7000-10000 unique glycopeptide precursors per compartment, per age. These findings are unprecedented in brain O-glycoproteomics. The age-related differences in glycosylation across brain regions provide valuable insights into dynamic changes during brain development and aging. Our high throughput glycoproteomics platform represents a significant advancement in glycoproteomic analysis, marking a major shift in research approaches for disease studies and biomarker discovery, leading to transformative progress in glycobiology and biomedical research.