In-depth site-specific analysis of N-glycoproteome in human cerebrospinal fluid (CSF) and glycosylation landscape changes in Alzheimer's disease (AD)
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Lingjun Li
PROVIDER: MSV000086395 | MassIVE | Sun Nov 01 13:25:00 GMT 2020
SECONDARY ACCESSION(S): PXD022274
REPOSITORIES: MassIVE
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Project description:Site-specific O-glycoproteome mapping in complex biological system provides molecular basis for understanding the structure-function relationships of glycoproteins and their roles in physiological and pathological processes. Previous O-glycoproteome analysis in cerebrospinal fluid (CSF) focused on sialylated glycoforms, and many CSF glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. In order to provide an unbiased O-glycosylation profiling, we have developed an integrated strategy combining universal boronic acid enrichment, high-pH fractionation, and electron-transfer and higher-energy collision dissociation (EThcD) for improved intact O-glycopeptide analysis. This strategy was applied to analyze O-glycoproteome in CSF, leading to identifications of 308 O-glycopeptides from 110 O-glycoproteins, covering both sialylated and non-sialylated glycoforms. To our knowledge, this is the largest number of O-glycoproteins and O-glycosites reported for CSF so far, including 154 novel O-glycosites. Due to a lack of peptidomics workflow that could incorporate glycosylation analysis, the glycosylation state of CSF endogenous peptides has not been comprehensively studied. Here, we developed a peptidomics workflow that utilizes the EThcD fragmentation and a three-step database searching strategy, allowing both N-glycosylation and O-glycosylation, as well as other common peptide PTMs, to be analyzed at the same time. Interestingly, among the 1492 endogenous peptides identified, 95 of them were O-glycosylated and only 1 N-glycosylated peptide was found, indicating CSF endogenous peptides were preferentially O-glycosylated. By referring to human neuropeptide database, 15 of them were actually O-glycosylated neuropeptides deriving from ProSAAS and secretogranin-1. O-glycoproteome and endogenous peptidome PTMs analysis were also conducted in MCI and AD patients to give a landscape picture of glycosylation in these disease states. The results showed that a decreased fucosylation trend was found in MCI and AD, suggesting its potential relation to the progression of AD.
2021-04-05 | MSV000087160 | MassIVE
Project description:GalNAc-transferase (GalNAc-T) isoforms modify distinct subsets of the O-glycoproteome and GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and T2) in liver cells, and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and T2. Differential transcriptomic analysis indicates that loss of function of a GalNAc-T induces specific transcriptional response. The non-redundant O-glycoproteome subsets for and the transcriptional responses for each isoform appeared to be related to different cellular processes, and for the GalNAc-T2 isoform supporting a role in lipid metabolism. The results demonstrate that GalNAc-Ts have non-redundant glycosylation functions, and that these may affect distinct cellular processes. The data provides a comprehensive resource for unique substrates for individual GalNAc-Ts. Our study provides a new view on the regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
2015-09-30 | E-MTAB-3844 | biostudies-arrayexpress
Project description:We demonstrate the utility of lectin-magnetic nanoprobe coupled with Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. Using non-small cell lung cancer (NSCLC) cells as model, this approach allowed us to compare the glycosylation profiles of the drug resistant cell, PC9, and its resistant derivative, PC9-IR. The complementary enrichment allowed broader targeting of different glycoforms; using Byonic software, confident identification of a total of 2767 and 2290 non-redundant intact glycopeptides was achieved for PC9-IR and PC9, respectively. This also enabled glycan profiling of the onco-protein EGFR, without immunoprecipitation. Moreover, the glycan specificity afforded by the lectin@MNP allowed as to observe increased terminal and core fucosylation in EGFR from PC9-IR, which demonstrates the potential for protein glycosylation alterations in the resistant cell line. The multi-lectin nanoprobe-based affinity mass spectrometry presents a new method for large-scale glycoproteomic profiling of different sample types without prior fractionation or immunoprecipitation.
2022-02-25 | PXD010035 | Pride
Project description:BackgroundAlthough Amyloid-beta and Tau are the hallmarks of Alzheimer's Disease (AD), other protein pathways such as endothelial dysfunction may be involved and may precede cognitive symptoms. Our objective was to characterize the cerebrospinal fluid (CSF) proteomic profiles focusing on cardiometabolic-related protein pathways in individuals on the AD spectrum.MethodsWe performed CSF and plasma-targeted proteomics (276 proteins) from 354 participants of the Brain Stress Hypertension and Aging Program (BSHARP), of which 8% had preclinical AD, and 24% had MCI due to AD. We instituted a bioinformatic pipeline to generate data-driven protein modules, used "Hub" and "Critical" proteins within each module to describe protein signatures for each AD stage and then assessed their associations with clinical and biological AD traits. Finally, we completed pathway enrichment analysis to get insight into pathways that might be implicated in AD pathogenesis.ResultsThe 276 measured proteins clustered into five modules that were associated with CSF Amyloid-β42, Tau, and pTau. (all p-value <0.05). A CSF protein AD signature was characterized by elevated levels of CSF Hepatocyte Growth Factor (HGF), Intercellular and Vascular Cell Adhesion Molecule 1 (ICAM-1, VCAM-1), Neuropilin 1 and 2 (NRP-1, NRP-2), Scavenger Receptor Class B Member 2(SCARB2), Plasminogen Activator, Urokinase (PLAU). (all <0.05) We also found a significant difference in the CSF/Plasma ratio for the proteins associated with Cognitive Status and the Tau/Aβ42 ratio (TAR) in the CSF. Pathway enrichment analysis revealed that cell adhesion and endothelial dysfunction (all p-value <0.05) were key mechanisms involved in AD pathogenesis, especially in the preclinical stage.ConclusionOur results suggest a proteomic signature in the CSF of individuals with preclinical AD that is driven by adhesion molecules and might be implicated in the pathogenesis of AD. Future studies investigating these pathways may provide insights into novel AD biomarkers and therapeutic targets.
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Project description:Protein glycosylation is a highly diverse post-translational modification, modulating key cellular processes such as cell signaling, adhesion and cell-cell interactions. Its deregulation has been associated with various pathologies, including cancer and neurological diseases. Methods capable of quantifying glycosylation dynamics are essential to start unraveling the biological functions of protein glycosylation. Here we present Deep Quantitative Glycoprofiling (DQGlyco), a method that combines high-throughput sample preparation, high-sensitivity detection, and precise multiplexed quantification of protein glycosylation. We used DQGlyco to profile the mouse brain glycoproteome, in which we identify more than 200,000 unique N-glycopeptides - this amounts to 25-fold more glycopeptides identified compared to previous studies. We observed extensive heterogeneity of glycoforms and determined their functional and structural preferences. We used our quantitative approach to map surface-exposed glycoforms as well as to characterize glycosites tissue-specificity. The presence of a defined gut microbiota resulted in extensive remodeling of the brain glycoproteome when compared to that of germ-free animals, exemplifying how the gut microbiome may affect brain protein functions.
2025-02-07 | PXD052447 | Pride
Project description:Alterations of protein abundance and post-translational modifications in patients with Alzheimer’s disease (AD), such as glycosylation, phosphorylation, and ubiquitination, and their roles in disease progression and treatment outcome are areas of intense study. Little is known, however, about the overall N-glycosylation of proteins in human brains, and those from Alzheimer’s patients, particularly in regard to large-scale intact N-linked glycoproteomics analysis. To elucidate the glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography (HILIC), and LC-MS-based glycoproteomics analysis. We analyzed 10 normal, 10 asymptomatic, and 10 symptomatic AD brains, in which we detected >300 glycoproteins and >1,900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic and complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain glycoproteome, occurring at <9% of N-glycans . We observed changes in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among normal, asymptomatic, and symptomatic AD samples. Further analysis revealed glycosylation differences in subcellular compartments. We did not observe a statistical difference between male and female patients. These results represent the first glycoproteomics landscape of brains from multiple AD individuals, which will facilitate a deeper understanding of AD and possible disease treatment options.
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