SPE-CZE-MS Quantifies Zeptomole Amounts of Phosphorylated Peptides

ABSTRACT: Capillary zone electrophoresis (CZE) is gaining attention in the field of single-cell proteomics for its ultra-low-flow and high-resolution separation abilities. Even more sample-limited yet rich in biological information are phosphoproteomics experiments, as the phosphoproteome composes only a fraction of the whole cellular proteome and preparation for this kind of analysis requires an additional enrichment step. Rapid analysis, high sensitivity, and maximization of sample utilization is paramount for single-cell analysis. Some challenges of coupling CZE analysis with mass spectrometry analysis (MS) of complex mixtures include 1. sensitivity due to volume loading limitations of CZE and 2. incompatibility of MS duty cycles with electropherographic timescales, especially for rapid (<20 min) analyses. Here, we address these two challenges as applied to single-cell equivalent phosphoproteomics experiments by interfacing a microchip-based CZE device integrated with a solid-phase-extraction (SPE) bed with the Orbitrap Astral mass spectrometer. The SPE bed theoretically circumvents the volume limitations that thwart detection sensitivity of classical CZE by allowing analyte capture and concentration before electrokinetic focusing and separation. Further, we leverage the high sampling rates of the Orbitrap Astral mass spectrometer to take full advantage of the fast and high-resolution SPE-CZE separations which historically pose challenges for typical MS scan speeds. Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS analysis. We characterize important device characteristics such as volume and mass loading tolerances as well as compatibility with state-of-the-art mass spectrometry methods. Finally, we highlight unique SPE-CZE separation mechanisms that can empower fit-for-purpose applications in single-cell-equivalent phosphoproteomics.

INSTRUMENT(S): Orbitrap Astral

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Joshua Coon  

PROVIDER: MSV000096595 | MassIVE | Wed Dec 04 18:14:00 GMT 2024

SECONDARY ACCESSION(S): PXD058591

REPOSITORIES: MassIVE