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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Astral, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: Karl Mechtler  

PROVIDER: MSV000097281 | MassIVE | Mon Mar 10 14:29:00 GMT 2025

SECONDARY ACCESSION(S): PXD049412

REPOSITORIES: MassIVE