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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 (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Epithelial Cell, Stem Cell

DISEASE(S): Lung Cancer

SUBMITTER: Manuel Matzinger

LAB HEAD: Karl Mechtler

PROVIDER: PXD049412 | Pride | 2025-01-16

REPOSITORIES: pride

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			Action	DRS
	20230910_250pgHeLa_240k_20Th_40ms_DDM_2.raw	Raw
	20230910_250pgHeLa_240k_20Th_40ms_DDM_3.raw	Raw
	20230910_250pgHeLa_240k_20Th_40ms_DDM_4.raw	Raw
	20231010_10ngHeLa_240k_5Th_10ms_DDM_50SPD_1.raw	Raw
	20231010_10ngHeLa_240k_5Th_10ms_DDM_50SPD_1.raw_1_RunOverview.tsv	Raw

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Project description:Genes and signaling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analyzed. Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE the expression of NANOG, SOX2 and POU5F1 was indeed increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was affected. The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent but epiblast-like state. These data indicate that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells. Microarrays used were bovine whole genome gene expression microarrays V2 (Agilent Technologies) representing 43,653 Bos taurus 60-mer oligos in a 4x44K layout. RNA samples from morula, blastocyst and dissected inner cell mass (ICM) and trophectoderm (TE) were compared in a common reference experiment design using 8 dual channel microarrays with each sample hybridized against an identical sample consisting of a pool of blastocysts total RNA. Within each group of two microarrays for each stage/tissue type, sample versus common reference hybridizations were performed in balanced dye-swap.

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

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