Recent advances in LC-MS have enabled label-free single cell proteome analysis revealing unexpected functional diversity of cells. However, there are still key challenges in this field application such as sensitivity, coverage, dynamic range, and throughput. To address some of these challenges, new method developments, as well optimization on existing LC-MS-based proteomics workflows are necessary. Here, we demonstrate the use of Orbitrap Ascend mass spectrometer, nano- UHPLC and solid silicon micro-pillar array column technology for high-throughput single cell applications.
Individual HeLa cells were isolated using CellenONE system followed by reduction, alkylation and trypsin digestion as per manufacturer’s instructions and reconstituted in 0.1% TFA prior to LC-MS analysis. Pierce™ HeLa digest was used for dilution series from 50 pg to 5 ng loaded on column . Single cell digests and the diluted standard HeLa digest samples were analyzed using Orbitrap Ascend mass spectrometer FAIMS Pro™ interface coupled to a Vanquish Neo UHPLC system. Separation was performed on a 50 cm µPAC™ Neo column. Data was acquired in a DIA mode and searched with a beta version of Thermo Scientific Proteome Discoverer Software 3.1.
The performance of this ultra-sensitive LC-MS workflow was first optimized using a dilution series of HeLa digest. From 250 pg of HeLa digest load, we could identify on average > 3,000 protein groups by library-free approach whereas using DIA-library generated from 10 ng HeLa digest, we were able to identify >5,000 protein groups using a method with a throughput of 80 samples per day.