Data-Independent\nAcquisition Shortens the Analytical\nWindow of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis\nMass Spectrometry

Separation\nin single-cell mass spectrometry (MS) improves molecular\ncoverage and quantification; however, it also elongates measurements,\nthus limiting analytical throughput to study large populations of\ncells. Here, we advance the speed of bottom-up proteomics by capillary\nelectrophoresis (CE) high-resolution mass spectrometry (MS) for single-cell\nproteomics. We adjust the applied electrophoresis potential to readily\ncontrol the duration of electrophoresis. On the HeLa proteome standard,\nshorter separation times curbed proteome detection using data-dependent\nacquisition (DDA) but not data-independent acquisition (DIA) on an\nOrbitrap analyzer. This DIA method identified 1161 proteins vs 401\nproteins by the reference DDA within a 15 min effective separation\nfrom single HeLa-cell-equivalent (∼200 pg) proteome digests.\nLabel-free quantification found these exclusively DIA-identified proteins\nin the lower domain of the concentration range, revealing sensitivity\nimprovement. The approach also significantly advanced the reproducibility\nof quantification, where ∼76% of the DIA-quantified proteins\nhad <20% coefficient of variation vs ∼43% by DDA. As a proof\nof principle, the method allowed us to quantify 1242 proteins in subcellular\nniches in a single, neural-tissue fated cell in the live Xenopus laevis (frog) embryo, including many canonical\ncomponents of organelles. DIA integration enhanced throughput by ∼2–4\nfold and sensitivity by a factor of ∼3 in single-cell (subcellular)\nCE-MS proteomics.