Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

McClatchy, Daniel B.; Liao, Lujian; Park, Sung Kyu; Venable, John D.; Yates, John R.

doi:10.1101/gr.6375007

Cited by 100 publications

(115 citation statements)

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“…Steps 2-11 had a similar profile with the following changes: 5 min in 100% (vol/vol) buffer A, 3 min in X% (vol/vol) buffer C, a 10-min gradient from 0 to 15% (vol/vol) buffer B, and a 108-min gradient from 15-100% (vol/vol) buffer B. The 3-min buffer C percentages (X) were 10,20,30,40,50,60,70,80,90, and 100% (vol/vol), respectively, for the 11-step analysis. As peptides eluted from the microcapillary column, they were electrosprayed directly into an LTQ Velos Orbitrap mass spectrometer (Thermo Finnigan) with the application of a distal 2.4-kV spray voltage.…”

Section: Methodsmentioning

confidence: 91%

“…15 N labeling of rodents facilitates accurate, proteome-wide quantitative analysis of long-lived proteins and synapse-wide changes during development (19)(20)(21). Out of more than 7,000 quantified proteins, we identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, and we validated 22 of these proteins by immunoblotting and one by immunohistochemistry.…”

Section: Significancementioning

confidence: 99%

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In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Butko¹,

Savas

Friedman³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high-and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensorydeprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. mass spectrometric proteomics | somatosensory cortex | experience-dependent plasticity | synaptic protein dynamics S ensory information is encoded in the brain by activation of specific neuronal circuits that operate via chemical synapses. Important sensory experiences are stored by strengthening activated synapses in the circuit, a process known as "experience-dependent plasticity" (1). When animals are deprived of sensory experience during development, for example, by trimming rodent whiskers from birth to 30 d after birth, synaptic strength and mature synaptic morphology are attenuated, suggesting that the low activity in the deprived circuits interferes with normal development (2-7). However, the molecular changes that alter these synaptic properties in response to experience remain unclear. Synaptic activation has been shown to promote regulated and highly localized effects on synaptic proteins such as local translation, recruitment, and targeted degradation (8-11), and this spatiotemporal control of protein availability is required for long-term plasticity and synaptic maturation, which are fundamental in memory formation and storage and ultimately for an organism's survival (12-14). The need for highly controlled protein availability is highlighted in studies of neurological diseases, which often result from the disruption of protein availability at the synapse (15-17). ...

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Section: Methodsmentioning

confidence: 91%

Section: Significancementioning

confidence: 99%

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Butko¹,

Savas

Friedman³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Stable isotope labeling in mammals (SILAM) enables complete labeling of amino acids with 15 N in the whole animal, including rodents (22,23). This method has been successfully applied to analyze synaptic proteome dynamics in developing brain (24), to quantify neuronal proteome phosphorylation events in neurons (25), and to study protein turnover and identify long-lived proteins in neuronal nuclear pore complexes (26). To further differentiate the role of de novo translation in protein changes from alterations in other processes such as mRNA stability, protein degradation, or trafficking, stable isotope pulsed labeling (pSILAC) has been developed to measure genome-scale protein synthesis rates (27).…”

mentioning

confidence: 99%

Fmr1 deficiency promotes age-dependent alterations in the cortical synaptic proteome

Tang

Wang

Wan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by severe intellectual disability and other symptoms including autism. Although caused by the silencing of a single gene, Fmr1 (fragile X mental retardation 1), the complexity of FXS pathogenesis is amplified because the encoded protein, FMRP, regulates the activity-dependent translation of numerous mRNAs. Although the mRNAs that associate with FMRP have been extensively studied, little is known regarding the proteins whose expression levels are altered, directly or indirectly, by loss of FMRP during brain development. Here we systematically measured protein expression in neocortical synaptic fractions from Fmr1 knockout (KO) and wild-type (WT) mice at both adolescent and adult stages. Although hundreds of proteins are up-regulated in the absence of FMRP in young mice, this up-regulation is largely diminished in adulthood. Up-regulated proteins included previously unidentified as well as known targets involved in synapse formation and function and brain development and others linked to intellectual disability and autism. Comparison with putative FMRP target mRNAs and autism susceptibility genes revealed substantial overlap, consistent with the idea that the autism endophenotype of FXS is due to a "multiple hit" effect of FMRP loss, particularly within the PSD95 interactome. Through studies of de novo protein synthesis in primary cortical neurons from KO and WT mice, we found that neurons lacking FMRP produce nascent proteins at higher rates, many of which are synaptic proteins and encoded by FMRP target mRNAs. Our results provide a greatly expanded view of protein changes in FXS and identify age-dependent effects of FMRP in shaping the neuronal proteome.fragile X | quantitative mass spectrometry | synaptic protein synthesis | autism | stable isotope labeling F ragile X syndrome (FXS) is an X-linked monogenic disorder that leads to highly debilitating changes in neurodevelopment. Affected individuals exhibit mental retardation, attention deficit and hyperactivity, anxiety, autism spectrum behaviors, and other symptoms that compound overall impairment (1). In the vast majority of cases, FXS is caused by an mRNA-dependent epigenetic silencing of the Fmr1 (fragile X mental retardation 1) gene (2), which occurs secondarily to a CGG repeat expansion in the 5′ UTR region of Fmr1 (3) and results in absence of the encoded protein FMRP. FMRP is an RNA-binding protein that regulates several aspects of mRNA translation (1), transport (4), and stability (5) in neurons. Substantial evidence indicates that FMRP is particularly critical as a suppressor of activity-dependent mRNA translation at glutamatergic synapses (6, 7) and that loss of this function results in abnormalities in dendritic spine shape and several forms of long-term synaptic plasticity (8, 9). In addition, significant changes have been described regarding the structure and/or function of other synaptic systems, including GABAergic and endocannabinoid synapses (10, 11). Loss of FMR...

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“…Precipitated P. falciparum protein preparations were dissolved in digestion buffer, digested with trypsin and LysC, and analyzed by LC/LC/MS/MS according to published protocols [22,23]. Approximately 100 mg of protein was used for a 6-step for soluble samples and 12-step for insoluble LC/LC/MS/MS analysis on a LTQ-Orbitrap, a hybrid mass spectrometer in which a linear ion trap is coupled to an Orbitrap mass analyzer (ThermoElectron, San Jose, CA).…”

Section: Multidimensional Protein Identification Technology (Mudpit)mentioning

confidence: 99%

Large-Scale Differential Proteome Analysis in Plasmodium falciparum Under Drug Treatment

Prieto

Fischer

Koncarevic³

et al. 2014

Methods in Molecular Biology

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Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using 14 N-isoleucine and 13 C 6 , 15 N 1 -isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (.1.5) whereas 14 and 8 proteins were down-regulated (,0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 mg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites.

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Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

Cited by 100 publications

References 83 publications

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

Fmr1 deficiency promotes age-dependent alterations in the cortical synaptic proteome

Large-Scale Differential Proteome Analysis in Plasmodium falciparum Under Drug Treatment

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