Electrophoresis-Correlative Data-Independent Acquisition\n(Eco-DIA) Improves the Sensitivity of Mass Spectrometry for Limited\nProteome Amounts

Capillary zone electrophoresis (CE)\ncombines high separation power,\nscalability, and speed to limited proteome analyses by mass spectrometry\n(MS). However, compressed separation in CE challenges the duty cycle\nof tandem MS, even during data-independent acquisition (DIA). To help\nremedy this limitation, we introduce the concept of <u>e</u>lectrophoresis-<u>co</u>rrelative (Eco) data acquisition\nfor CE-MS. We recognize CE electrospray ionization (ESI) to sort peptide\nions into reproducible mass-to-charge (<i>m</i>/<i>z</i>) vs migration time (MT) trends in the solution phase,\nbefore subsequent ionization and <i>m</i>/<i>z</i> analysis. We proposed that such a correlation can be leveraged to\nimprove the economy of data acquisition. We test this hypothesis using\nDIA frames that are tailored to the observed <i>m</i>/<i>z</i>–MT trends. The resulting Eco-DIA method substantially\nimproves the bandwidth utilization of tandem MS during CE-MS. In proof-of-principle\nstudies, Eco-DIA identified and quantified ∼38% more proteins\nfrom 1 ng of the HeLa proteome digest compared to the classical DIA,\nwithout the assistance of a project-specific tandem MS spectral library.\nEco-DIA was able to quantify ∼51% more proteins with <10%\ncoefficient of variation vs the control DIA approach. Based on label-free\nquantification, the proteins that were exclusively measured by Eco-MS\noccupied the lower dynamic range of the detected proteome concentration,\nrevealing sensitivity enhancement. In addition to marking the inception\nof Eco-MS, this work lays the foundation for the development of next-generation\ndata acquisition strategies that leverage electrophoretic ion sorting\nfor high-sensitivity proteomics.