Introduction
To understand and characterize cell heterogeneity individual cells need to be studied. In recent years single cell analysis has profited from advances in LC-MS based proteomics approaches and tools. Nevertheless, there are still challenges in this field of application. Besides sample preparation, the key challenges in looking at individual single cell proteomes are sensitivity, coverage, dynamic range, and throughput. To address some of these challenges, new technological developments, as well as improvements on existing LC-MS-based proteomics workflows are a necessity. Here, we evaluated the performance of the Orbitrap Astral mass spectrometer for high-throughput single cell applications.
Methods
HeLa cells were isolated and prepared using a CellenONE® system. Pierce™ HeLa digest (10 ug) was reconstituted by adding 100 μL of 0.015% DDM solution, sonicated for 5 min, then diluted in 0.015% DDM solution to 5 ng/μL. Single cell digests and the diluted standard HeLa digest samples were analyzed using the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer with Thermo Scientific™ FAIMS Pro™ interface coupled to a Thermo Scientific™ Vanquish™ Neo UHPLC system. Data was acquired in a DIA mode and processed with Spectronaut 18 software.
Preliminary Data
Using a 80 SPD method and 250 pg HeLa digest we could identify >5,000 protein groups and >25,000 peptides using a library-free search, and >6,600 protein groups and >42,000 peptides using a 3 x 10 ng HeLa library search. Using 50 SPD method and 250 pg Pierce HeLa digest we could identify >6,100 protein groups and >41,000 peptides using a library-free search, and >7,700 protein groups and >66,000 peptides using a 3 x 10 ng HeLa library search. Median CV values at protein groups level were always <10%. The analysis of HeLa single cell samples resulted in ~4900 protein groups and 25,000 peptides using library-free search and ~5600 protein groups and 32,000 peptides using a 3 x 20 HeLa cells library search.