In a study focused on single-cell proteomics, researchers aimed to overcome limitations related to proteomic depth and quantitative performance. They achieved this by using a combination of a zero dead-end volume chromatographic column and the Thermo Scientific Orbitrap Astral mass spectrometer in data-independent acquisition (DIA) mode. This approach allowed them to achieve an unprecedented depth of proteome coverage and accurate quantification, even at ultra-low input amounts.
Using a tailored library, the researchers identified up to 7,400 protein groups from as little as 250 pg of HeLa cells at a throughput of 50 samples per day. They also benchmarked different data analysis strategies, demonstrating that false discovery rates (FDR) on the protein level can be maintained at 1%. Additionally, they successfully determined fold change differences of 2 at single-cell input levels.
The workflow was applied to A549 cells, resulting in the identification of up to 5,200 protein groups from a single cell, enabling the study of cellular heterogeneity and dependencies between cell size and cell-cycle phase. The researchers also used the workflow to distinguish between in vitro analogs of two human blastocyst lineages.
Overall, this study represents a significant advancement in single-cell proteomics, offering improved sensitivity, reproducibility, and quantitative accuracy, making it a valuable tool for biologists to complement single-cell genomics data.
The study utilized an Aurora Ultimate TS 25 cm nanoflow UHPLC, enabling the achievement of unprecedented proteome coverage and accuracy in quantifying ultra-low input amounts in single-cell proteomics.
J. A. Bubis; T. N. Arrey; E Damoc; B. Delanghe; J Slovakova; T M. Sommer; H. Kagawa,; P Pichler; N. Rivron, K. Mechtler, M. Matzinger
Challenging the Astral mass analyzer – going beyond 5200 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.