In-depth site-specific analysis of N-glycoproteome in human cerebrospinal fluid (CSF) and glycosylation landscape changes in Alzheimer's disease (AD)
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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: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.
2022-10-31 | PXD032219 | Pride
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:Alzheimer’s disease (AD) is an age-related neurodegenerative disorder in which neuroinflammation plays a critical function. Recurring viral infections and loss of immune competence increase risk for developing AD, yet the cellular and molecular mechanisms driving these immune changes are unknown. Here we performed mass cytometry of peripheral blood mononuclear cells and detected an immunologic signature of AD characterized by increased numbers of CD8+ T effector memory CD45RA+ (TEMRA) cells. CD8+ TEMRA cells were negatively associated with cognition and single cell RNA sequencing revealed their cytotoxic effector function. Strikingly, we discovered identical, shared T cell receptors (TCRs) of clonally expanded CD8+ TEMRA cells in cerebrospinal fluid (CSF) of three AD patients. Deep TCR sequencing, machine learning, and peptide screens identified the HLA-B*08:01-restricted Epstein-Barr virus trans-activator protein BZLF1 as the cognate antigen of a novel AD CSF TCR . These results provide the first evidence of clonal, antigen-specific T cells patrolling the intrathecal space of brains affected by age-related neurodegeneration
2019-11-04 | GSE134577 | GEO
Project description:Abnormal insulin signaling in the brain has been linked to Alzheimer's disease (AD).To evaluate whether cerebrospinal fluid (CSF) insulin levels are associated with cognitive performance and CSF amyloid-? and Tau. Additionally, we explore whether any such association differs by sex or APOE ?4 genotype.From 258 individuals participating in the Parelsnoer Institute Neurodegenerative Diseases, a nationwide multicenter memory clinic population, we selected 138 individuals (mean age 66±9 years, 65.2% male) diagnosed with subjective cognitive impairment (n?=?45), amnestic mild cognitive impairment (n?=?44), or AD (n?=?49), who completed a neuropsychological assessment, including tests of global cognition and memory performance, and who underwent lumbar puncture. We measured CSF levels of insulin, amyloid-?1-42, total (t-)Tau, and phosphorylated (p-)Tau.CSF insulin levels did not differ between the diagnostic groups (p?=?0.136). Across the whole study population, CSF insulin was unrelated to cognitive performance and CSF biomarkers of AD, after adjustment for age, sex, body mass index, diabetes status, and clinic site (all p?0.131). Importantly, however, we observed effect modification by sex and APOE ?4 genotype. Specifically, among women, higher insulin levels in the CSF were associated with worse global cognition (standardized regression coefficient -0.483; p?=?0.008) and higher p-Tau levels (0.353; p?=?0.040). Among non-carriers of the APOE ?4 allele, higher CSF insulin was associated with higher t-Tau (0.287; p?=?0.008) and p-Tau (0.246; p?=?0.029).Our findings provide further evidence for a relationship between brain insulin signaling and AD pathology. It also highlights the need to consider sex and APOE ?4 genotype when assessing the role of insulin.
| S-EPMC5734123 | biostudies-literature
Project description:BackgroundAlzheimer's disease (AD) cerebrospinal fluid (CSF) biomarker cutoffs from immunoassays with low interlaboratory variability in diverse ethnic groups are necessary for their use in clinics and clinical trials. With lack of cutoffs from fully automated immunoassay platforms in diverse races, the aim of this study is to evaluate the clinical utility of CSF AD biomarkers from the Lumipulse fully automated immunoassay based on β-amyloid (Aβ) positron emission tomography (PET) status comparing with these from two manual immunoassays, in Koreans.MethodsAmong 331 Korean participants enrolled from a prospective, 3-year longitudinal observational study of the validation cohort of Korean Brain Aging Study for the Early Diagnosis and Prediction of AD, 139 (29 CN, 58 SCD, 29 MCI, and 23 AD) provided CSF and 271 underwent baseline amyloid PET (n = 128 with overlapping CSF and Aβ-PET, and 143 without CSFs). Three annual cognitive and neuropsychiatric function tests were conducted. Aβ42, Aβ40, total-tau, and phosphorylated-tau181 were measured by Lumipulse fully automated immunoassay and two manual immunoassays (INNO-BIA AlzBio3, INNOTEST). Clinical utility of CSF biomarker cutoffs, based on 128 participants with Aβ-PET, was evaluated.ResultsCognitive and neuropsychological scores differed significantly among the groups, with descending performance among CN>SCD>MCI>AD. Biomarker levels among immunoassays were strongly intercorrelated. We determined the Aβ-PET status in a subgroup without CSF (n = 143), and then when we applied CSF biomarker cutoffs determined based on the Aβ-PET status, the CSF biomarkers (cutoffs of 642.1 pg/mL for Aβ42, 0.060 for Aβ42/Aβ40, 0.315 for t-tau/Aβ42, and 0.051 for p-tau/Aβ42, respectively) showed good agreement with Aβ-PET (overall AUC ranges of 0.840-0.898). Use of the Aβ-PET-based CSF cutoffs showed excellent diagnostic discrimination between AD and CN (Aβ42, Aβ42/Aβ40, t-tau/Aβ42, and p-tau/Aβ42) with overall AUC ranges of 0.876-0.952. During follow-up, participants with AD-like CSF signature determined by Aβ-PET-based cutoffs from Lumipulse showed rapid progression of cognitive decline in 139 subjects, after adjustment for potential confounders, compared with those with a normal CSF signature.ConclusionCSF AD biomarkers measured by different immunoassay platforms show strong intercorrelated agreement with Aβ-PET in Koreans. The Korean-specific Aβ-PET-based CSF biomarker cutoffs measured by the Lumipulse assay strongly predicts progression of cognitive decline. The clinical utility of CSF biomarkers from fully-automated immunoassay platforms should be evaluated in larger, more diverse cohorts.
| S-EPMC7802266 | biostudies-literature
Project description:BackgroundLittle is known of vitamin D concentration in cerebrospinal fluid (CSF) in Alzheimer's disease (AD) and its relation with CSF acetylcholinesterase (AChE) activity, a marker of cholinergic function.MethodsA cross-sectional study of 52 consecutive patients under primary evaluation of cognitive impairment and 17 healthy controls. The patients had AD dementia or mild cognitive impairment (MCI) diagnosed with AD dementia upon follow-up (n = 28), other dementias (n = 12), and stable MCI (SMCI, n = 12). We determined serum and CSF concentrations of calcium, parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), and CSF activities of AChE and butyrylcholinesterase (BuChE).FindingsCSF 25OHD level was reduced in AD patients (P < 0.05), and CSF AChE activity was decreased both in patients with AD (P < 0.05) and other dementias (P < 0.01) compared to healthy controls. None of the measured variables differed between BuChE K-variant genotypes whereas the participants that were homozygous in terms of the apolipoprotein E (APOE) ?4 allele had decreased CSF AChE activity compared to subjects lacking the APOE ?4 allele (P = 0.01). In AD patients (n=28), CSF AChE activity correlated positively with CSF levels of total tau (T-tau) (r = 0.44, P < 0.05) and phosphorylated tau protein (P-tau) (r = 0.50, P < 0.01), but CSF activities of AChE or BuChE did not correlate with serum or CSF levels of 25OHD.ConclusionsIn this pilot study, both CSF 25OHD level and CSF AChE activity were reduced in AD patients. However, the lack of correlations between 25OHD levels and CSF activities of AChE or BuChE might suggest different mechanisms of action, which could have implications for treatment trials.
| S-EPMC3843721 | biostudies-literature
Project description:As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.
| S-EPMC8724636 | biostudies-literature
Project description:Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and the most common form of dementia in the elderly. Susceptibility to AD is considerably determined by genetic factors which hitherto were primarily identified using case-control designs. Elucidating the genetic architecture of additional AD-related phenotypic traits, ideally those linked to the underlying disease process, holds great promise in gaining deeper insights into the genetic basis of AD and in developing better clinical prediction models. To this end, we generated genome-wide single-nucleotide polymorphism (SNP) genotyping data in 931 participants of the European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) sample to search for novel genetic determinants of AD biomarker variability. Specifically, we performed genome-wide association study (GWAS) analyses on 16 traits, including 14 measures derived from quantifications of five separate amyloid-beta (Aβ) and tau-protein species in the cerebrospinal fluid (CSF). In addition to confirming the well-established effects of apolipoprotein E (APOE) on diagnostic outcome and phenotypes related to Aβ42, we detected novel potential signals in the zinc finger homeobox 3 (ZFHX3) for CSF-Aβ38 and CSF-Aβ40 levels, and confirmed the previously described sex-specific association between SNPs in geminin coiled-coil domain containing (GMNC) and CSF-tau. Utilizing the results from independent case-control AD GWAS to construct polygenic risk scores (PRS) revealed that AD risk variants only explain a small fraction of CSF biomarker variability. In conclusion, our study represents a detailed first account of GWAS analyses on CSF-Aβ and -tau-related traits in the EMIF-AD MBD dataset. In subsequent work, we will utilize the genomics data generated here in GWAS of other AD-relevant clinical outcomes ascertained in this unique dataset.
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