PXD049412-1

PXD049412 is an original dataset announced via ProteomeXchange.

Title	Challenging the Astral mass analyzer – up to 5300 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.
Description	A detailed proteome map is crucial for understanding molecular pathways and protein functions. Despite significant advancements in sample preparation, instrumentation, and data analysis, single-cell proteomics is currently limited by proteomic depth and quantitative performance. We combine a zero dead-end volume chromatographic column running at high throughput with the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer running in DIA mode. We yield unprecedented depth of proteome coverage as well as accuracy and precision for quantification of ultra-low input amounts. Using a tailored library, we identify up to 7400 protein groups from as little as 250 pg HeLa at a throughput of 50 samples per day (SPD). We benchmark multiple data analysis strategies, estimate their influence on FDR and show that FDR on protein level can easily be maintained at 1 %. Using a two-proteome mix, we check for optimal parameters of quantification and show that fold change differences of 2 can still be successfully determined at single-cell level inputs. Eventually, we apply our workflow to A549 cells yielding a proteome coverage of up to 5200 protein groups from a single cell, which allows the observation of heterogeneity in a cellular population and studying dependencies between cell size and cell-cycle phase. Additionally, our workflow enables us to distinguish between in vitro analogs of two human blastocyst lineages: naïve human pluripotent stem cells (epiblast) and trophectoderm (TE)-like cells. Gene Ontology analysis of enriched proteins in TE-like cells harmoniously aligns with transcriptomic data, indicating that single-cell proteomics possesses the capability to identify biologically relevant differences between these two lineages within the blastocyst.
HostingRepository	PRIDE
AnnounceDate	2025-01-16
AnnouncementXML	Submission_2025-01-16_13:55:45.317.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Manuel Matzinger
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606; scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Astral; Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2024-02-15 12:39:30	ID requested
⏵ 1	2025-01-16 13:55:45	announced

10.1038/S41592-024-02559-1;

submitter keyword: liquid chromatography, mass spectrometry, Astral, A549, Aurora column,single-cell proteomics, trophectoderm-like cells, naïve human stem cells

Karl Mechtler
contact affiliation	Research Institute of Molecular Pathology (IMP), Vienna-Bio-Center (VBC), Vienna, Austria
contact email	karl.mechtler@imp.ac.at
lab head
Manuel Matzinger
contact affiliation	IMP / University of Vienna
contact email	manuel.matzinger@imp.ac.at
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/2025/01/PXD049412

PRIDE project URI

• PRIDE
  1. PXD049412
     1. Label: PRIDE project
     2. Name: Challenging the Astral mass analyzer – up to 5300 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.