Electrophoresis-Correlative diaPASEF Improves Low-Input and Single-Cell CE−ESI−MS Proteomics by Targeting Peptide-Enriched Acquisition Space

Abstract

In low-input proteomics, proteome coverage is often limited not only by sample amount but also by inefficient tandem mass spectrometry (MS) sampling of peptide-rich acquisition space. During capillary electrophoresis (CE)−electrospray ionization (ESI)−MS analysis, electrophoretically separated peptides occupy reproducible, correlated regions of migration time, precursor mass-to-charge ratio (m/z), and ion mobility (IM), creating an opportunity to direct data-independent acquisition (DIA) toward peptide-enriched regions. Here, we introduce electrophoresis-correlative diaPASEF (Eco-diaPASEF), a CE-informed implementation of diaPASEF that uses migration time-correlated m/z and IM information to define complementary acquisition segments while maintaining the reference cycle time. By restricting IM scan range, precursor m/z space, and DIA isolation width, Eco-diaPASEF focuses tandem MS acquisition on peptide-enriched regions. In low-input HeLa digest samples, Eco-diaPASEF increased protein-group (PG) identifications by up to ∼65% relative to conventional diaPASEF, cumulatively identifying up to 1163 PGs across 4 technical replicates from 200 pg of digest measured per run on a timsTOF Pro mass spectrometer. Applied to single animal-cap cells from stage-9 Xenopus laevis embryos, Eco-diaPASEF cumulatively quantified 1108 PGs across 3 cells using ∼200 pg of injected digest per cell. The method provided ∼2.6−2.7-fold greater proteome coverage across replicate criteria while maintaining comparable biological-replicate precision. These results establish Eco-diaPASEF as a CE-guided acquisition strategy for improving proteome coverage in low-input and single-cell CE−ESI−MS proteomics on IM-enabled mass spectrometers.