Julia Kraegenbring (Bremen / DE), Fernanda Salvato (San Jose, CA / US), Bernard Delanghe (Bremen / DE), David Hartlmayr (Lyon / FR), Anjali Seth (Lyon / FR), Heiner Koch (Bremen / DE), Amirmansoor Hakimi (San Jose, CA / US)
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 Exploris 480 mass spectrometer and the Vanquish Neo UHPLC system for high-throughput single cell applications.
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 Exploris 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.
The performance of this ultra-sensitive LC-MS workflow was optimized using a dilution series of HeLa digest. From 250 pg of HeLa digest load, we could identify on average > 2,000 protein groups by library-free approach whereas using DIA-library generated from 5 ng HeLa digest, we were able to identify >3,000 protein groups using a method with a throughput of 50 samples per day.