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23rd Human Proteome Organization World Congress

Discovering novel oligo and polysialylated proteins

Keynote speaker

Keynote lecture

Prof.

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

Glycobiology Insights

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&amp;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.

Oral presentation

Prof. Dr.

Finding the sweet spot – the pursuit of single cell glycomics by CE-MS

IntroductionThere is still an urgent clinical need for new specific molecular targets that can be utilized to establish better diagnostic, prognostic as well as therapeutic treatments for cancer patients. A hallmark of many diseases, including cancer, are alterations in glycosylation (e.g. covalent attachment of sugar moieties to proteins or lipids), providing new opportunities for better patient stratification. This is not surprising as glycosylation is involved in many biological processes, and glycan structures cover our cells in a dense layer and contribute to the malignant phenotype of cancer cells by promoting proliferation, metastasis, and immunosuppression. However, a more thorough investigation is needed on the glycomic intratumoral heterogeneity (e.g. primary versus metastatic tumors), as tumors consist of a network of cell populations that are interconnected in tissue matrix. Therefore, it is of utmost importance that analytical workflows are used that can distinguish between these populations, and reveal the relevant cells by single cell analysis. However, current glycomic methods still require a large amount of cells (20,000 up to 100,000 cells). Here, we present an innovative and miniaturized high-sensitivity glycomics workflow (capillary electrophoresis coupled to mass spectrometry; CE-MS).MethodsVarious conditions of the 96-well polyvinylidene difluoride (PVDF) membrane approach were evaluated (e.g. membrane blocking and N-glycan release conditions). By implementing the release on the membrane as well as using a hydrazide labeling procedure, no additional purification steps were required. Analysis was performed on a CE-MS platform and the complete workflow was further evaluated by its application for the characterization of the N-glycome from total plasma, extracellular vesicles and two cell lines (PaTu-S and JY) using various amount of lysed cells (1 – 200,000 cells).ResultsA highly diverse N-glycome was observed for all samples and the CE-MS demonstrated its ability to separate isomeric structures, in particular differently linked sialic acids, without the need of an additional derivatization step. A total of 72 N-glycans could be relatively quantified from 5,000 PaTu-S cells using the optimized protocol. The five most abundant N-glycans could still be detected using ~1 cell after cell lysis.ConclusionsIn this study we achieved an exceptional sensitivity for glycomic analysis that only requires minimal sample amounts (e.g., up to a single cell or 100 pg of protein weight). Future endeavors will focus on using the workflow for isolated single cells (sorted by the CellenONE system) as well as to apply it on laser-capture microdissected samples. In time, this will allow us to gain in-depth insights into the role of glycosylation in tumor progression and how these alterations can be exploited for personalized treatment strategies through improved diagnosis and prognosis of cancer patients.

New insights into the lysosomal glycoproteome

Lysosomes, essential catabolic cellular organelles packed with diverse lysosomal hydrolases, play critical roles in biomolecular degradation, autophagic processes and extracellular cell-cell communication. While the protein complement of the lysosome is well-studied, the lysosomal glycoproteome remain poorly understood despite the recognised importance of protein glycosylation including mannose-6-phosphorylation (M6P) in lysosome biology. To this end, we performed an in-depth glycoprofiling of both soluble and membrane protein fractions from isolated liver lysosomes (tritosomes) from wild-type (WT, n = 3-5) and acid-phosphatase 2/5 double knockout (ACP2/5-dKO, n = 3) mice using quantitative glycomics and glycoproteomics. ACP2/5-dKO mice were included in the study to also map the unprocessed lysosomal glycoproteome ahead of the final M6P removal from trafficking lysosomal glycoproteins. From WT lysosomes, we identified 1000+ unique N-glycopeptides covering 200+ N-glycosylation sites from 120 source glycoproteins comprising the expected lysosomal hydrolases. Glycomics identified 60 different N-glycan isomers spanning a surprisingly high abundance of paucimannose (Man1-3GlcNAc2Fuc0-1, &gt;50% of the N-glycome) in addition to oligomannosidic and complex-type N-glycans. As expected, no M6P-tagged glycoproteins were found in the WT lysosomes. The WT lysosome displayed different N-glycome profiles across the soluble and membrane protein fractions indicating differential N-glycan processing of those protein subsets. Surprisingly, the glyco(proteo)mics data of ACP2/5-dKO lysosomes revealed very low levels of M6P (&lt;10% of the N-glycoproteome) on only a few soluble lysosomal proteins (prosaposin, cathepsin D/L, dipeptidyl-peptidase-1). Similar to the WT lysosomes, the ACP2/5-dKO lysosomes instead displayed a paucimannose-rich glycophenotype (~50% of the N-glycome) and only relatively little oligomannosylation and complex-type N-glycosylation indicating that only a small proportion of the lysosomal hydrolases traffic to mouse liver lysosomes via the well-established M6P-tagging pathway. This study generates, for the first time, a comprehensive and quantitative N-glycoproteome map of the mouse liver lysosome representing an important resource to further unravel the glycobiology of lysosomes and their trafficking pathways.

Plenary hall

Session

Glycoproteomics

Session
Glycoproteomics

Termin

Vorsitzende

Programm

SessionGlycoproteomics

Termin

Vorsitzende

Programm

Session
Glycoproteomics