The 15-min (Sub)Cellular Proteome.

Single-cell mass spectrometry (MS) opens a proteomic window onto the inner workings of cells. Here, we report the discovery characterization of the subcellular proteome of single, identified embryonic cells in record speed and molecular coverage. We integrated subcellular capillary microsampling, fast capillary electrophoresis (CE), high-efficiency nano-flow electrospray ionization, and orbitrap tandem MS. In proof-of-principle tests, we found shorter separation times to hinder proteome detection using DDA, but not DIA. Within a 15-min effective separation window, CE data-independent acquisition (DIA) was able to identify 1,161 proteins from single HeLa-cell-equivalent (∼200 pg) proteome digests vs. 401 proteins by the reference data-dependent acquisition (DDA) on the same platform. The approach measured 1,242 proteins from subcellular niches in an identified cell in the live Xenopus laevis (frog) embryo, including many canonical components of organelles. CE-MS with DIA enables fast, sensitive, and deep profiling of the (sub)cellular proteome, expanding the bioanalytical toolbox of cell biology. Authorship Contributions:P.N. and B.S. designed the study. L.R.P. collected the X. laevis cell aspirates. B.S. prepared and measured the samples. B.S. and P.N. analyzed the data and interpreted the results. P.N. and B.S. wrote the manuscript. All the authors commented on the manuscript.