Dataset Information

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

ABSTRACT: N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

SUBMITTER: Fang P

PROVIDER: S-EPMC5122430 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

Fang Pan P Wang Xin-Jian XJ Xue Yu Y Liu Ming-Qi MQ Zeng Wen-Feng WF Zhang Yang Y Zhang Lei L Gao Xing X Yan Guo-Quan GQ Yao Jun J Shen Hua-Li HL Yang Peng-Yuan PY

Oncotarget 20160601 25

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation st ...[more]

PMID: 27259237

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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.

Project description:Neurons in the mouse auditory cortex are strongly influenced by behavior, including both suppression and enhancement of sound-evoked responses during movement. The mouse auditory cortex comprises multiple fields with different roles in sound processing and distinct connectivity to movement-related centers of the brain. Here, we asked whether movement-related modulation might differ across auditory cortical fields, thereby contributing to the heterogeneity of movement-related modulation at the single-cell level. We used wide-field calcium imaging to identify distinct cortical fields followed by cellular-resolution two-photon calcium imaging to visualize the activity of layer 2/3 excitatory neurons within each field. We measured each neuron's responses to three sound categories (pure tones, chirps, and amplitude modulated white noise) as mice rested and ran on a non-motorized treadmill. We found that individual neurons in each cortical field typically respond to just one sound category. Some neurons are only active during rest and others during locomotion, and those that are responsive across conditions retain their sound-category tuning. The effects of locomotion on sound-evoked responses vary at the single cell level, with both suppression and enhancement of neural responses, and the net modulatory effect of locomotion is largely conserved across cortical fields. Movement-related modulation in auditory cortex also reflects more complex behavioral patterns, including instantaneous running speed and non-locomotor movements such as grooming and postural adjustments, with similar patterns seen across all auditory cortical fields. Our findings underscore the complexity of movement-related modulation throughout the mouse auditory cortex and indicate that movement related modulation is a widespread phenomenon.Significance statementThroughout the sensory cortex, neural activity is influenced by behavior. It remains unknown whether primary and higher-order sensory cortical centers are similarly or differentially influenced by movement. We show that movement-related modulation in the mouse auditory cortex is locally complex and heterogeneous, but that at a more macroscopic level, the net effect of movement on primary and higher-order auditory cortex is largely conserved. These data highlight the widespread nature of movement-related modulation and suggest that movement signals may inform neural computations throughout multiple nodes of the sensory cortex.

Dataset Information

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

Publications

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

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