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Proteomics of MCF7 cells (LC-MS/MS Orbitrap Fusion Tribrid)

ABSTRACT: We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. We compared riboLace translation estimates with protein levels determined by proteomics in MCF7 cells. RiboLace can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Toma Tebaldi

LAB HEAD: Gabriella Viero

PROVIDER: PXD009417 | Pride | 2018-11-21

REPOSITORIES: Pride

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			Action	DRS
	MCF7_tot_1.raw	Raw
	MCF7_tot_2.raw	Raw
	MCF7_tot_3.raw	Raw
	MaxQuant_andromeda.zip	Other
	MaxQuant_txt.zip	Other

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Publications

Active Ribosome Profiling with RiboLace.

Clamer Massimiliano M Tebaldi Toma T Lauria Fabio F Bernabò Paola P Gómez-Biagi Rodolfo F RF Marchioretto Marta M Kandala Divya T DT Minati Luca L Perenthaler Elena E Gubert Daniele D Pasquardini Laura L Guella Graziano G Groen Ewout J N EJN Gillingwater Thomas H TH Quattrone Alessandro A Viero Gabriella G

Cell reports 20181001 4

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possi ...[more]

PMID: 30355487

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Active translatome profiling with RiboLace in MCF7 cells

Project description:We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, it works with very low input material and can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems. Keywords: translation, RiboLace, polysome profiling, RNA-seq, POL-seq, active translation

2018-10-09 | GSE112295 | GEO

Active ribosome profiling with RiboLace and standard ribosome profiling in HEK-293 cells

Project description:We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, it works with very low input material and can be easily and rapidly used to enhance Ribo-Seq, obtaining a global snapshot on active ribosome footprints at single nucleotide resolution from eukaryotic in vitro and in vivo systems. Keywords: ribosome profiling, translation, RiboLace, Ribo-Seq, active translation

2018-10-09 | GSE112353 | GEO

Active ribosome profiling from mouse brains with RiboLace

2018-10-09 | GSE102354 | GEO

A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation [Ribo-Seq]

Project description:We used Ribo-seq to determine how PRMT inhibition affects translation of different mRNAs, This analysis can generate a “snapshot” of all mRNAs that are occupied by active ribosomes (i.e., undergoing active translation) in a cell at a particular condition.

2022-06-30 | GSE153142 | GEO

Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3M-bM-^@M-^Y UTRs

Project description:The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3M-bM-^@M-^Y untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3M-bM-^@M-^Y UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Examination of Upf1 binding preferences via iCLIP in untreated HeLa cells and HeLa cells, where translation is blocked by puromycin treatment in vivo crosslinking and immunoprecipitation strategy (iCLIP)

2013-06-20 | E-GEOD-47976 | biostudies-arrayexpress

De novo protein synthesis occurs through cytoplasmic translation machinery in mammalian spermatozoa

Project description:For decades, it was widely accepted that mammalian spermatozoa were devoid of translational activity due to the functional and morphological changes that occurred during spermatogenesis. However, it was proved that capacitated spermatozoa possess translation activity, which appears to be ensured by mitochondrial ribosomes. Since mitochondrial translation has several particularities, the hypothesis that sperm translation occurs through mitochondrial ribosomes raises some questions. The main objectives of this work were to study the impact of translation inhibition in the bovine spermatozoa proteome and to identify the de novo synthesized proteins by capacitated bovine spermatozoa. To identify which proteins were de novo translated, capacitated bovine spermatozoa were treated with puromycin and puromycin-peptides were immunoprecipitated. To study the impact of translation inhibitors in capacitated sperm proteome, bovine spermatozoa were capacitated in the presence or absence of translation inhibitors - Cycloheximide (CHX, cytoplasmic translation inhibitor) and D-Chloramphenicol (D-CP, mitochondrial translation inhibitor). After capacitation, sperm lysates were analysed by mass spectrometry. We identify nine newly synthesised cytoplasmic proteins by capacitated bovine spermatozoa, which are related to sperm-specific processes. Both translation inhibitors impact differently the sperm proteome leading to the deregulation of cytoplasmic protein related to sperm physiology and function. These results support the existence of cytoplasmic translation in bovine spermatozoa during capacitation.

2025-06-13 | PXD050868 | Pride

Capture, release, and identification of newly synthesized proteins for improved profiling of functional translatomes

Project description:Proteins undergoing active translation in proliferating K562 cells during a 2-hour time frame were labeled with O-propargyl-puromycin for subsequent click reaction with cleavable Dde biotin-azide. Following capture on streptavidin agarose resin, nascent polypeptides were released from the resin by treatment with 2% hydrazine. The method was used to profile the nascent proteome upon acute inhibition of mTOR with MLN128, an ATP-competitive active-site inhibitor of the mTOR kinase.

2022-07-22 | MSV000089950 | MassIVE

Snapshot of translation in mammalian cells that are depleted of polyamines or replete with polyamines

Project description:Snapshot of translation in mammalian cells that are depleted of polyamines or replete with polyamines. Hek293T cells treated with DFMO or Spermidine.

2018-10-11 | GSE111517 | GEO

Change of the mRNA level upon Puromycin treatment

Project description:Change in the levels of mRNAs upon Puromycin treatment were examined to analyze the mRNA population(s) affected directly or indirectly by global translation

2025-07-21 | GSE85794 | GEO

SPECtre: a spectral coherence-based classifier of actively translated transcripts from ribosome profiling sequence data

Project description:Active protein translation can be assessed and measured using ribosome profiling sequencing strategies. Existing approaches make use of sequence fragment length or frame occupancy to differentiate between active translation and background noise, however they do not consider additional characteristics inherent to the technology which limits their overall accuracy. Here, we present an analytical tool that models the overall tri-nucleotide periodicity of ribosomal occupancy using a classifier based on spectral coherence. Our software, SPECtre, examines the relationship of normalized ribosome profiling read coverage over a rolling series of windows along a transcript against an idealized reference signal. A comparison of SPECtre against current methods on existing and new data shows a marked improvement in accuracy for detecting active translation and exhibits overall high sensitivity at a low false discovery rate. Classification of actively translated transcripts in ribosome profiling data derived from human neuroblastoma SH-SY5Y cells, and data previously published derived from mouse embryonic stem cells and zebrafish embryos.

2016-07-01 | E-GEOD-75947 | biostudies-arrayexpress