CE-guided diaPASEF acquisition raises protein identifications from picogram and single-cell samples

Summary. Researchers at the University of Maryland reported in Analytical Chemistry, published online on 20 October, electrophoresis-correlative diaPASEF (Eco-diaPASEF), a data-acquisition strategy that improves protein coverage in low-input and single-cell proteomics by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). In low-input work, coverage is limited not only by the amount of sample but also by inefficient tandem mass spectrometry sampling of the regions where peptides actually appear. During CE-ESI-MS, separated peptides occupy reproducible, correlated regions of migration time, mass-to-charge ratio and ion mobility.

Research context (RUO). Eco-diaPASEF uses these correlations to direct data-independent acquisition (DIA) toward peptide-rich regions. Based on migration time, the method defines complementary acquisition segments that restrict the ion-mobility scan range, the precursor mass-to-charge window and the DIA isolation width, while keeping the reference cycle time. In low-input HeLa cell-line digest samples analysed on a timsTOF Pro mass spectrometer, Eco-diaPASEF increased protein-group identifications by up to about 65 percent compared with conventional diaPASEF, cumulatively identifying up to 1163 protein groups across four technical replicates from 200 picograms of digest per run. Applied to single animal-cap cells from stage-9 embryos of the frog Xenopus laevis, it cumulatively quantified 1108 protein groups across three cells, with about 200 picograms of digest analysed per cell. The method gave about 2.6 to 2.7 times greater proteome coverage across replicate criteria while keeping comparable precision between biological replicates. The authors present Eco-diaPASEF as a CE-guided acquisition strategy for ion-mobility-enabled mass spectrometers. The cell-line digest serves only as an analytical test sample, and the paper is an analytical-methods report with no clinical component. For mass-spectrometry laboratories, it shows how separation-aware acquisition windows can recover depth when sample amounts are extremely small.