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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Dataset's files

Source:

			Action	DRS
	230420_12_Orbi3_SK_SER_G60_T85_TTY_Koppel_9ul_DMSO_1.raw	Raw
	230420_14_Orbi3_SK_SER_G60_T85_TTY_Koppel_9ul_OPP_2.raw	Raw
	230420_16_Orbi3_SK_SER_G60_T85_TTY_Koppel_9ul_OPP_PAT_1.raw	Raw
	230420_18_Orbi3_SK_SER_G60_T85_TTY_Koppel_9ul_DMSO_2.raw	Raw
	230420_20_Orbi3_SK_SER_G60_T85_TTY_Koppel_9ul_OPP_1.raw	Raw

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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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