MassIVE MSV000097281

Imported Reanalysis Dataset Public PXD049412

Challenging the Astral mass analyzer – up to 5300 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.

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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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Liquid chromatography ; Mass spectrometry ; Astral ; A549 ; Aurora column ; Single-cell proteomics ; Trophectoderm-like cells ; Naïve human stem cells ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Karl Mechtler, Research Institute of Molecular Pathology (IMP), Vienna-Bio-Center (VBC), Vienna, Austria, N/A
Submitting User: ccms
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Experimental Design
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Identification Results
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Quantification Results
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When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.