Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry

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