Electrophoresis-Correlative Framing of Ion Mobility Deepens Single-Cell Mass Spectrometry Proteomics

This project developed electrophoresis-correlative (Eco) mass spectrometry (MS) to measure the proteome of single cells on a trapped ion mobility mass spectrometer (timsTOF PRO). Eco-MS identified 962 protein groups in a <20 min of electrophoresing a single-HeLa-cell-equivalent (~200 pg) proteome amount. Match-between-runs improved this number to 2,139 protein groups on the same dataset. We demonstrate the approach to measure the proteome of single cells in X. laevis embryos at stage 8 (blastula). Single cells were isolated from the animal cap and their proteomes digested on a fluorosilane-coated microplate. Less than 4% of the available single-cell proteome was analyzed on a custom-built capillary electrophoresis (CE) platform under classical and Eco-driven ddaPASEF. Eco-MS returned ~51% of more protein groups than the control, identifying 1,157 proteins from N = 9 different single cells (biological replicates) of the animal cap. Eco-MS deepens the detectable coverage of the single-cell proteome using ddaPASEF on a timsTOF MS.