Dataset Information

Puromycin acetyltransferase-mediated cell-specific labeling of de novo synthesized proteins, Part II

ABSTRACT: Puromycin and its derivative O-propargyl puromycin (OPP) enable detection of nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here we have used puromycin N-acetyltransferase (PAT), for cell-specific metabolic labelling with puromycin or OPP. This approach, which we named Puromycin Inactivation for Cell-Selective proteome Labelling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing PAT and detection of translation in other cell types. We performed two mass spectrometry experiments using cell-specific expression of PAT, metabolic labeling of cells with OPP and streptavidin pulldown of biotinylated de novo synthesized proteins. In the first proof-of-principle experiment (Part I) we mixed human (HEK293) and mouse (Neuro2a) cells and labeled them with OPP, comparing this with another sample where HEK293 cells expressed PAT. We show specific reduction of human-specific peptides in de novo synthesized proteome in HEK-PAT cells. In the second experiment (Part II), we used a primary co-culture of rat cortical neurons (expressing PAT in some samples) and glia, performed OPP labeling and pulldown of de novo synthesized proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Rattus Rattus (black Rat)

SUBMITTER: Indrek Koppel

LAB HEAD: Indrek Koppel

PROVIDER: PXD043498 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

Cell type-specific labeling of newly synthesized proteins by puromycin inactivation.

Cabrera-Cabrera Florencia F Tull Helena H Capuana Roberta R Kasvandik Sergo S Timmusk Tõnis T Koppel Indrek I

The Journal of biological chemistry 20230804 9

Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here, we show how a widely used mammalian selection marker, puromycin N-acetyltransferase, can be repurposed for cell-specific metabolic labeling. This approach, which we named puromycin inactivation for cell-selective proteome labeling (P ...[more]

PMID: 37543363

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Project description:Neuronal networks are subject to fluctuations in both the magnitude and frequency of inputs, requiring plasticity mechanisms to stabilize network activity. Homeostatic synaptic scaling is a form of synaptic plasticity that adjusts the strength of neuronal connections up or down in response to large changes in input. Although homeostatic plasticity requires changes in gene expression, there is only limited data describing the molecular changes associated with homeostatic scaling, focusing mostly on the expression mechanisms involving glutamate receptors. The fact that neuronal networks can be scaled up (in response to reduced activity) or down (in response to enhanced activity) provides a unique opportunity to examine the molecular and proteomic response to opposite ends of the phenotypic spectrum of synaptic plasticity. Here we first demonstrate that homeostatic scaling required protein synthesis. We then examined the plasticity-induced changes in the newly-synthesized neuronal proteome of neurons to identify the landscape of proteomic changes that contribute to opposing forms of homeostatic plasticity. Cultured rat hippocampal neurons (21 DIV) underwent homeostatic upscaling or downscaling (treatments with TTX and Bicucculine, respectively). We used BONCAT (BioOrthogonal Non-Canonical Amino acid Tagging) to metabolically label, capture and identify newly-synthesized proteins, detecting and analysing 5940 newly-synthesized proteins using liquid chromatography-coupled tandem mass spectrometry and label-free quantitation. Neither up- or down-scaling produced changes in the number of different proteins translated. Rather, our findings indicate that synaptic up- and down-scaling elicit opposing translational regulation of several molecular pathways, producing targeted adjustments in the neuronal proteome. We detected ~ 300 differentially regulated proteins involved in neurite outgrowth, reorganization of nerve terminals, axon guidance and targeting, neurotransmitter transport, filopodia assembly, excitatory synapses and glutamate receptor complexes. These proteins include well-characterized mediators of synaptic plasticity, e.g. the ionotropic glutamate receptor complex that is down-regulated during down-scaling and coordinately upregulated during upscaling. We also identified differentially regulated proteins that in addition to their regulation in homeostatic plasticity, are also associated with multiple diseases and disorders, including intellectual disability, schizophrenia, epilepsy, and Parkinson’s disease.

Dataset Information

Puromycin acetyltransferase-mediated cell-specific labeling of de novo synthesized proteins, Part II

Publications

Cell type-specific labeling of newly synthesized proteins by puromycin inactivation.

Similar Datasets

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