Background

Recent advances in LC-MS have enabled label-free single-cell proteome analysis, revealing unexpected functional diversity in cells. However, there are still key challenges in this field, such as sensitivity, coverage, dynamic range, and throughput. To address some of these challenges, new method developments as well as optimization of existing LC-MS-based proteomics workflows are necessary. Here, we demonstrate the use of the Orbitrap Ascend Tribrid mass spectrometer and the Vanquish Neo UHPLC system for high-throughput single cell applications.

Methods

Individual HeLa cells were sorted, followed by reduction, alkylation, and trypsin digestion using CellenONE as per the manufacturer"s protocols. Pierce HeLa digest was used for dilution series from 50 pg to 10 ng loaded on column. Single cell digests and the diluted standard HeLa digest samples were analyzed using the Orbitrap Ascend Tribrid MS with the FAIMS Pro interface coupled to the Vanquish Neo UHPLC system. Separation was performed on the Aurora Ultimate TS 25cm column. Data was acquired in a DIA mode and searched with a beta version of Proteome Discoverer Software 3.1 and Spectronaut 18.

Results

The performance of this ultra-sensitive LC-MS workflow was first optimized using a dilution series of HeLa digests. From 250 pg of HeLa digest load, we could identify on average 3,200 protein groups by a library-free approach, whereas using DIA-library generated from 5 and 10 ng of HeLa digest, we were able to identify around 5,000 protein groups using a method with a throughput of 50 samples per day. HeLa single cells resulted in an average of 3,700 protein groups and 21,000 peptides using library-free searches and 4,400 protein groups and >26,000 peptides in library-based searches.

Conclusion

End-to-end workflow for single-cell proteomics on an Orbitrap Ascend Tribrid MS with sensitive detections of proteins from single cell or low-input samples.