PXD056651

PXD056651 is an original dataset announced via ProteomeXchange.

Title	A human tri-partite cell-free translation system
Description	Understanding which factors confer specific functions in any molecular process is fundamental to understanding the system as a whole. Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate, particularly in developmental models. The proteome constitutes the ultimate manifestation of gene expression programs; thus, multiple layers of regulation exist to ensure the faithful execution of such programs. While current high-throughput techniques aimed at investigating regulation at the level of translation, such as ribosome profiling and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential biases and confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have remained rigid and often required artificially designed mRNA constructs to probe for specialized mechanisms. Here, we detail a human tri-partite cell-free translation system based on the efficient separation of ribosomes, ribosome-depleted cytoplasmic lysate, and mRNAs, allowing for flexible reconstitution of translation reactions. In this setup, some translation components, such as the cytoplasmic lysate, can be kept constant while others, such as ribosome complexes or mRNA complements, can be varied or be subjected to various treatments. We detail several steps and conditions that allow for the translation of complete mRNA populations as input and the detection of nascent peptides from such mRNA populations using mass spectrometry. We utilized this protocol to resolve which components of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which hitherto have been unresolvable. Finally, we describe how lysates can be prepared from genetically manipulated cells and efficiently and robustly incorporated into high-throughput drug screening pipelines.
HostingRepository	PRIDE
AnnounceDate	2025-04-16
AnnouncementXML	Submission_2025-04-16_12:16:08.553.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Frederic Arendrup
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	timsTOF Pro 2

Revision	Datetime	Status	ChangeLog Entry
0	2024-10-09 04:30:06	ID requested
⏵ 1	2025-04-16 12:16:08	announced

10.1038/S41596-025-01155-7;

submitter keyword: Human, ribosomes., HEK293

Anders H. Lund
contact affiliation	BRIC, University of Copenhagen, Denmark.
contact email	Anders.lund@bric.ku.dk
lab head
Frederic Arendrup
contact affiliation	University of Copenhagen
contact email	Frederic.arendrup@bric.ku.dk
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/04/PXD056651

PRIDE project URI

• PRIDE
  1. PXD056651
     1. Label: PRIDE project
     2. Name: A human tri-partite cell-free translation system